Biophotonic compositions, kits and methods

ABSTRACT

The present disclosure provides biophotonic topical compositions, kits and their uses. In particular, the biophotonic compositions of the present disclosure are substantially resistant to leaching such that very low amounts of chromophore(s) present in the biophotonic composition leach out of the composition. The biophotonic compositions and their uses are useful for promoting wound healing and skin rejuvenation, as well as treating acne and various skin disorders.

This application is a United States National Stage filing under 35U.S.C. § 371 of International Application No. PCT/CA2013/000395, filedApr. 19, 2013, which is a continuation-in-part of claims the benefit ofand is a continuation of U.S. patent application Ser. No. 13/830,488,filed Mar. 14, 2013, which claims the benefit of U.S. ProvisionalApplication Nos. 61/636,480, filed Apr. 20, 2012; 61/701,502, filed Sep.14, 2012; 61/636,574 filed on Apr. 20, 2012; 61/701,510, filed on Sep.14, 2012; 61/636,577, filed on Apr. 20, 2012; 61/701,513, filed on Sep.14, 2012; and 61/766,611, filed on Feb. 19, 2013; which applications arehereby incorporated by reference in their entireties. PCT/CA2013/000395also claims priority to U.S. Provisional Application Nos. 61/636,480,filed Apr. 20, 2012; 61/701,502, filed Sep. 14, 2012; 61/636,574 filedon Apr. 20, 2012; 61/701,510, filed on Sep. 14, 2012; 61/636,577, filedon Apr. 20, 2012; 61/701,513, filed on Sep. 14, 2012; and 61/766,611,filed on Feb. 19, 2013; which applications are hereby incorporated byreference in their entireties.

BACKGROUND

Phototherapy has recently been recognized as having wide range ofapplications in both the medical, cosmetic and dental fields for use insurgeries, therapies and examinations. For example, phototherapy hasbeen developed to treat cancers and tumors with lessened invasiveness.Phototherapy has also been used to disinfect target sites as anantimicrobial treatment. Phototherapy has also been found to promotewound healing.

Photodynamic therapy is a type of phototherapy which involves a step ofsystemic administration or uptake of a photosensitive agent into thediseased or injured tissue, which step is followed by site-specificapplication of activating light (photodynamic therapy). Such regimens,however, are often associated with undesired side-effects, includingsystemic or localized toxicity due to the direct contact of thephotosensitive agents with the tissues. Moreover, such existing regimensoften demonstrate low therapeutic efficacy due to, for example, the pooruptake of the photosensitive agents into the target tissues. Therefore,it is an object of the present disclosure to provide new and improvedcompositions and methods useful in phototherapy.

STATEMENT OF INVENTION

The present disclosure provides biophotonic compositions and methodsuseful in phototherapy. In particular, the biophotonic compositions ofthe present disclosure may comprise a chromophore in a medium, such as agelling agent, that provides a barrier such that the chromophore(s) andother components of the topical biophotonic compositions are not insubstantial contact with the target tissues, and/or do not penetrate thetarget tissues. Put another way, the biophotonic compositions of thepresent disclosure may contain a chromophore in a medium, such as agelling agent, which provides a barrier rendering the compositionssubstantially resistant to leaching in use. The use of such biophotoniccompositions in phototherapy would therefore not involve substantialdirect contact of the target tissues with a chromophore, which may bepotentially toxic the tissues or may cause undesired side effects.

From one aspect, there is provided a biophotonic composition comprisinga first chromophore; and a gelling agent present in an amount sufficientto gel the composition and render the biophotonic compositionsubstantially resistant to leaching such that less than 15% by weight ofthe total chromophore amount leaches out of the biophotonic compositionin use.

From one aspect, there is provided a biophotonic composition comprisinga first chromophore; and a gelling agent present in an amount sufficientto gel the composition and render the biophotonic compositionsubstantially resistant to leaching such that less than 15% by weight ofthe total chromophore amount leaches out of the biophotonic compositionin use, as measured by (i) placing a 2 mm thick layer of the biophotoniccomposition onto a top surface of a 2.4-3 cm diameter polycarbonate (PC)membrane with a thickness of 10 microns and a pore size of 3 microns,(ii) contacting a bottom surface of the PC membrane with a phosphatesaline buffer solution contained in a receptor compartment, and (iii)after a treatment time at room temperature and pressure, measuring thechromophore content in the receptor compartment.

From another aspect, there is provided a biophotonic compositioncomprising at least a first chromophore and a gelling agent, wherein thebiophotonic composition is a gel or a semi-solid and is substantiallyresistant to leaching such that less than 15% of the total chromophoreamount leaches out of the biophotonic composition into tissue when incontact with tissue in use. In certain embodiments, the biophotoniccomposition is spreadable so that it can conform to a tissue topography.

From yet another aspect, there is provided a biophotonic compositioncomprising at least a first chromophore and a gelling agent, wherein thebiophotonic composition is substantially translucent and issubstantially resistant to leaching such that less than 15% of the totalchromophore amount leaches out of the biophotonic composition intotissue when in contact with tissue in use. By substantially translucentis meant having a transmission of more than about 20%.

From a further aspect, there is provided a biophotonic compositioncomprising at least a first chromophore and a gelling agent, wherein thebiophotonic composition and/or the gelling agent has a viscosity ofabout 10,000-100,000, 10,000-90,000, 10,000-80,000, 10,000-70,000,15,000-80,000, 15,000-70,000, 15,000-50,000, or 15,000-45,000 cP whenmeasured using a Wells-Brookfield HB cone/plate viscometer and a CP-51cone at room temperature at a rotational speed of 2 rpm and atorque>10%, or a Brookfield DV-II+Pro viscometer with a spindle of 7, at50 rpm, 1 minute.

From a yet further aspect, there is provided a biophotonic composition,comprising a first chromophore in a carrier medium, wherein thecomposition is encapsulated in a membrane which membrane limits leachingof the first chromophore such that less than 15% of the totalchromophore amount leaches out of the composition in use. In certainembodiments, the membrane is substantially translucent. The membrane canbe selected from a lipid, a polymer, gelatin, cellulose, andcyclodextrins. The composition can also comprise a dendrimer, such asincluding poly(propylene amine). The carrier medium can be a liquid. Itcan also be a gel or semi-solid.

From another aspect, there is provided a biophotonic compositioncomprising a first chromophore and a gelling agent, wherein theviscosity of the biophotonic composition is about 10,000 to about100,000 cP, preferably about 10,000 to about 60,000 cP, more preferablyabout 10,000 to about 50,000 cP. In certain embodiments, the firstchromophore is a fluorophore which can absorb and emit light from withinthe composition. Preferably, the biophotonic composition has aspreadable consistency.

From a yet further aspect, there is provided a biophotonic compositioncomprising a first chromophore and a second chromophore in a medium,wherein at least one of the first and second chromophores is afluorophore. The first chromophore can be Fluorescein, and the secondchromophore Eosin Y. The first chromophore can be Eosin Y and the secondchromophore one or more of Rose Bengal, Phloxine B and Erythrosine B.

From another aspect, there is provided a biophotonic compositioncomprising first and second chromophores in a medium, wherein the firstchromophore is a fluorophore, and wherein light emitted by the firstchromophore after photoactivation can photoactivate the secondchromophore. In some embodiments of the above two aspects, the medium isa gel or is gel-like. The medium can have a spreadable consistency.

By ‘in use’ is meant during a treatment time which can be up to about 5minutes, up to about 10 minutes, up to about 15 minutes, up to about 20minutes, up to about 25 minutes, or up to about 30 minutes. Thetreatment time may comprise the total length of time that thecomposition is in contact with tissues.

Substantially resistant to leaching can be understand to mean less than15% of the total chromophore amount leaching out of the biophotoniccomposition into a phosphate saline buffer solution contained in areceptor compartment, through a 2.4-3 cm diameter polycarbonate (PC)membrane with a thickness of 10 microns and a pore size of 3 microns,having a top side onto which a 2 mm thick layer of the biophotoniccomposition is placed for 5 minutes at room temperature and pressure,and a bottom side which is in direct contact with the phosphate salinebuffer solution. It will be understood that if the treatment time islonger than 5 minutes, the leaching test needs to be extended to thetreatment time.

In certain embodiments of any of the foregoing or following, thebiophotonic topical composition allows less than 30%, 25%, 20%, 15%,10%, 5%, 1%, 0.8%, 0.5% or 0.1%, or essentially none of said chromophorecontent to leach out of the biophotonic composition.

In certain embodiments of any of the foregoing or following, thebiophotonic composition is a topical composition. Preferably, thecomposition is a gel, semi-solid or viscous liquid, which can be spreadon to the treatment site. In some embodiments, the composition canremain on the treatment site when the treatment site is inverted ortilted during the treatment time.

In certain embodiments of any of the foregoing or following, thebiophotonic composition is substantially translucent and/or transparent.By substantially translucent is meant having a transmission of more thanabout 20%. In some embodiments, the translucency comprises at least 20%,30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or 100%transmittance of light through a 2 mm thick composition.

In certain embodiments of any of the foregoing or following, thecomposition and/or the gelling agent has a viscosity of about10,000-100,000, 10,000-90,000, 10,000-80,000, 10,000-70,000,15,000-80,000, 15,000-70,000, 10,000-50,000, 10,000-40,000,15,000-50,000 or 15,000-40,000 cP when measured using a Wells-BrookfieldHB cone/plate viscometer and a CP-51 cone at room temperature at arotational speed of 2 rpm and a torque>10%, or a Brookfield DV-II+Proviscometer with a spindle of 7, at 50 rpm, 1 minute.

In certain embodiments of any of the foregoing or following, the gellingagent is selected from the group of cross-linked polymers. The polymerscan be covalently or physically cross-linked. The gelling agent can beselected from at least one of a hydrophilic material, a hygroscopicmaterial or a hydrated polymer. The gelling agent can be polyanionic incharge character. In some embodiments, the gelling agent comprisescarboxylic functional groups, which may contain from 2 to 7 carbon atomsper functional group.

The gelling agent can be a synthetic polymer selected from the groupconsisting of vinyl polymers, polyoxyethylene-polyoxypropylenecopolymers, poly(ethylene oxide), acrylamide polymers and derivatives orsalts thereof. The gelling agent can be a vinyl polymer selected fromthe group of polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone or polyvinyl alcohol. The gelling agent can be a carboxyvinyl polymer or a carbomer obtained by polymerisation of acrylic acid.The carboxy vinyl polymer or carbomer can be crosslinked.

In certain embodiments, the gelling agent is a high molecular weight,cross-linked polyacrylic acid polymer having a viscosity in the range ofabout 10,000-100,000; 10,000-80,000; 15,000-80,000; 10,000-70,000;15,000-70,000; 15,000-40,000, 10,000-60,000; 10,000-50,000;10,000-40,000; 20,000-100,000; 25,000-90,000; 30,000-80,000;30,000-70,000; 30,000-60,000; 25,000-40,000 cP. The polymer can beselected from the group consisting of, but not limited to Carbopol® 940,Carbopol® 980, ETD 2020 NF, Carbopol® 1382 Polymer, 71G NF, 971P NF,974P NF, 980 NF, 981 NF, 5984 EP, ETF 2020 NF, ultrez 10 NF, ultrez 20,ultrez 21, 1342 NF, 934 NF, 934P NF, 940 NF, 941 NF.

In certain embodiments, the gelling agent is a polyacrylic acid polymercross-linked with alkyl acrylate or allyl pentaerythritol and is presentin an amount of about 0.05% to about 5% by weight of the finalcomposition, preferably about 0.5% to about 2% by weight of the finalcomposition.

In certain embodiments of any of the foregoing or following, the gellingagent comprises a protein-based polymer, which can be selected from atleast one of sodium hyaluronate, gelatin and collagen. The gelling agentcan be gelatin and be present in an amount of equal to or more thanabout 4% by weight of the final composition. The gelling agent can becollagen and be present in an amount equal to or more than about 5% byweight of the final composition.

In certain embodiments of any of the foregoing or following, the gellingagent comprises a polysaccharide, which can be selected from at leastone of starch, chitosan, chitin, agar, alginates, xanthan, carrageenan,guar gum, gellan gum, pectin, and locust bean gum. The gelling agent canbe present in an amount equal to or more than about 0.01% by weight ofthe final composition.

In certain embodiments of any of the foregoing or following, the gellingagent comprises at least one glycol. The glycol can be selected fromethylene glycol and propylene glycol. The ethylene glycol can bepolyethylene glycol.

In certain embodiments, the biophotonic composition can further comprisea humectant, such as glycerine. The biophotonic may further comprisehealing factors, preservatives, pH adjusters, chelators or the like.

In certain embodiments of any of the foregoing or following, thebiophotonic composition is encapsulated in a membrane, which may beimpermeable or breathable to allow permeation of gases but not liquids.The membrane may be translucent. The membrane may comprise a lipid, apolymer or gelatin.

In certain embodiments of any of the foregoing or following, thebiophotonic composition further comprises an oxygen-releasing agentwhich can be a peroxide or a peroxide-releasing agent or water. Theoxygen-releasing agent can be selected from hydrogen peroxide, carbamideperoxide, benzoyl peroxide, peroxy acid, alkali metal peroxides, alkalimetal percarbonates, peroxyacetic acid, and alkali metal perborates.

In certain embodiments of any of the foregoing or following, the firstchromophore can be in an aqueous or alcohol solution in the composition.The gelling agent and the chromophore solution can form a hydrocolloid.

In certain embodiments of any of the foregoing or following, the firstchromophore absorbs light at a wavelength of 200-600 nm, or 400-800 nm.In certain embodiments of any of the foregoing or following, the firstchromophore absorbs light at a wavelength in the range of the visiblespectrum. In some embodiments, the first chromophore is a fluorescentchromophore (fluorophore). The first chromophore can be a xanthene dye.The first chromophore can be selected from Eosin Y, Eosin B, ErythrosinB, Fluorescein, Rose Bengal and Phloxin B. The first chromophore can bepresent in an amount of about 0.001% to about 40% by weight of the totalcomposition, preferably about 0.005% to about 2% by weight of the totalcomposition, more preferably about 0.01% to about 2% by weight of thetotal composition.

In certain embodiments of any of the foregoing or following, thecomposition further comprises a second chromophore. The firstchromophore can have an emission spectrum that overlaps at least 5%,10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% with an absorption spectrum ofthe second chromophore. In some embodiments, the first chromophore ofthe biophotonic topical composition has an emission spectrum thatoverlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%,35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of thesecond chromophore when present.

In certain embodiments of any of the foregoing or following, the firstchromophore transfers energy to the second chromophore upon illuminationwith a light. Illumination of the biophotonic topical composition withlight causes a transfer of energy from the first chromophore to thesecond chromophore. In some embodiments, the second chromophore emitsfluorescence and/or generates reactive oxygen species after absorbingenergy from the first chromophore. At least one of the chromophores, forexample, the first chromophore, can photobleach during illumination withlight. At least one of the chromophores, for example, the firstchromophore can emit fluorescence upon illumination with light. Incertain embodiments, the biophotonic composition does not generate asubstantial amount of heat following illumination with light. In someembodiments, the energy emitted by the biophotonic composition does notcause tissue damage.

In certain embodiments of any of the foregoing or following, the secondchromophore can absorb light at a wavelength in the range of the visiblespectrum. In some embodiments, the second chromophore has an absorptionwavelength that is relatively longer than that of the first chromophore,for example, 10-100 nm, 20-80 nm, 25-70 nm, or 30-60 nm longer.

In certain embodiments of any of the foregoing or following, the firstchromophore is Eosin Y, and the second chromophore is one or moreselected from Fluorescein, Phloxine B and Erythrosine B. In certainembodiments of any of the foregoing or following, the first chromophoreis Fluorescein, and the second chromophore is Eosin Y. Optionally, athird chromophore may be present such as Rose Bengal. In otherembodiments, the first chromophore is Rose Bengal. In some embodiments,the biophotonic composition comprises Eosin and Fluorescein. In otherembodiments, the biophotonic composition comprises Eosin and RoseBengal. In other embodiments, the biophotonic composition comprisesFluorescein and Rose Bengal. In other embodiments, the biophotoniccomposition comprises Fluorescein and Rose Bengal.

The second chromophore can be present in an amount of about 0.0001% toabout 40% by weight of the total composition, preferably about 0.0001%to about 2% by weight of the total composition.

In certain embodiments of any of the foregoing or following, thecomposition comprises a third chromophore. The third chromophore can bea chlorophyll (e.g. chlorophyllin, chlorophyll a, chlorophyll b) orsaffron.

In certain embodiments of any of the foregoing or following, the pH ofthe composition is within the range 4.0 to 7.0, preferably in the rangeof 4.0 to 6.5, more preferably in the range of 4.0 to 5.0. The pH of thecomposition may also be within the range 6.0 to 8.0, preferably in therange 6.5 to 7.5.

In certain embodiments of any of the foregoing or following, thebiophotonic composition may be applied to or impregnated into a materialsuch as a pad, a dressing, a woven or non-woven fabric or the like. Theimpregnated material may be used as a mask (e.g. a face mask) or adressing.

In certain embodiments of any of the foregoing or following, thebiophotonic composition further comprises at least one waveguide withinor adjacent to the composition. The waveguide can be a particle, a fibreor a fibrillar network made of a material which can transmit and/or emitlight.

In certain embodiments of any of the foregoing or following, thecomposition does not comprise opaque particles, such as silica.

From a further aspect, there is provided a biophotonic composition, asdescribed above, for use in skin rejuvenation; for use in the treatmentof wounds; for use in the treatment or prevention of skin disorders(such as acne, psoriasis); for use in the treatment or prevention ofperiodontitis; for use in the treatment of acute inflammation; or foruse in the treatment of fungal, bacterial or viral infections.

From a yet further aspect, there is provided use of a biophotoniccomposition, as described above, for skin rejuvenation, for thetreatment of wounds; for the treatment or prevention of skin disorders(such as acne, psoriasis); for the treatment or prevention ofperiodontitis; for the treatment of acute inflammation; or for thetreatment of fungal, bacterial or viral infections.

In another aspect, there is provided a method for providing cosmetictreatment, comprising applying topically to skin a biophotoniccomposition, as defined above; and illuminating said biophotoniccomposition with light having a wavelength that overlaps with anabsorption spectrum of the first chromophore. The cosmetic treatment canpromote skin rejuvenation.

In a further aspect, there is provided a method for promoting woundhealing, comprising: applying topically to a wound a biophotoniccomposition as defined above; and illuminating said biophotoniccomposition with light having a wavelength that overlaps with anabsorption spectrum of the first chromophore. In certain embodiments ofthe method, the wound as described herein includes for example chronicor acute wounds, such as diabetic foot ulcers, pressure ulcers, venousulcers or amputations. In some embodiments of the method for providingbiophotonic therapy to a wound, the method promotes reduction of scartissue formation.

In a yet further aspect, there is provided a method for biophotonictreatment of a skin disorder, comprising applying topically abiophotonic composition as defined above to a target skin tissueafflicted with the skin disorder; and illuminating said biophotoniccomposition with light having a wavelength that overlaps with anabsorption spectrum of the first chromophore.

In a further aspect, there is provided a method for biophotonictreatment of acne, comprising: applying topically a biophotoniccomposition, as defined above, to target tissue, wherein the tissue isan acne lesion or an acne scar; and illuminating said biophotoniccomposition with light having a wavelength that overlaps with anabsorption spectrum of the first chromophore.

In another aspect, there is provided a method of biophotonic treatmentof periodontal disease, comprising: applying topically a biophotoniccomposition, as defined above, to a periodontal pocket; and illuminatingsaid biophotonic composition with light having a wavelength thatoverlaps with an absorption spectrum of the first chromophore.

In certain embodiments of any method of the present disclosure, thebiophotonic composition is illuminated for any time period per treatmentin which the biophotonic composition is activated, for example 1 to 30minutes, preferably less than 20 minutes, 15 minutes, 10 minutes orabout 5 minutes. The treatment time can correspond to, or be longer thana time it takes for the first chromophore to photobleach. In certainembodiments, the method of the present disclosure comprises a step ofilluminating the biophotonic composition for a period of at least 30seconds, 2 minutes, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, or 30 minutes. In some embodiments, thebiophotonic composition is illuminated for a period of at least 3minutes. Preferably, the biophotonic composition is illuminated withvisible non-coherent light, such as violet and/or blue light. Any othersuitable light source can be used.

The distance of the light source from the biophotonic composition can beany distance which can deliver an appropriate light power density to thebiophotonic composition and/or the skin tissue, for example 5, 10, 15 or20 cm. The biophotonic composition is applied topically at any suitablethickness. Typically, the biophotonic composition is applied topicallyto skin or wounds at a thickness of at least about 2 mm, about 2 mm toabout 10 mm.

In certain embodiments of the methods of the present disclosure, thebiophotonic composition is removed from the site of a treatmentfollowing application of light. Accordingly, the biophotonic compositionis removed from the site of treatment within at least 30 seconds, 2minutes, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes or 30 minutes after application. In someembodiments, the biophotonic composition is removed after a period of atleast 3 minutes post application of the biophotonic composition totreatment site.

In certain other embodiments, the composition remains in the treatmentarea and can be re-illuminated as required. The biophotonic compositioncan be kept in place for up to one, two or three weeks. The compositioncan be re-illuminated with light, which may include ambient light, atvarious intervals. In this case, the composition may be covered up inbetween exposure to light. For example, the biophotonic composition maybe soaked in a dressing and placed inside or over a wound and be left inplace for an extended period of time (e.g. more than one day).

In certain embodiments of the method for biophotonic treatment acne, thetreatment can be applied to the skin tissue, such as on the face, once,twice, three times, four times, five times or six times a week, daily,or at any other frequency. The total treatment time can be one week, twoweeks, three weeks, four weeks, five weeks, six weeks, seven weeks,eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, or anyother length of time deemed appropriate. In certain embodiments, theface may be split into separate areas (cheeks, forehead), and each areatreated separately. For example, the composition may be appliedtopically to a first portion, and that portion illuminated with light,and the biophotonic composition then removed. Then the composition isapplied to a second portion, illuminated and removed. Finally, thecomposition is applied to a third portion, illuminated and removed.

In certain embodiments of the method for biophotonic treatment ofwounds, the treatment can be applied in or on the wound once, twice,three times, four times, five times or six times a week, daily, or atany other frequency. The total treatment time can be one week, twoweeks, three weeks, four weeks, five weeks, six weeks, seven weeks,eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, or anyother length of time deemed appropriate.

The disclosed methods for treating acne, wounds or other skin conditionsmay further include, for example, administering a systemic or topicaldrug before, during or after the biophotonic treatment. The drug may bean antibiotic, a hormone treatment, or any other pharmaceuticalpreparation which may help to treat acne or wounds. The combination of asystemic treatment together with a topical biophotonic treatment canreduce the duration of systemic treatment time.

From another aspect there is provided a kit comprising a composition asdescribed above, and one or more of a light source for activating thechromophore, instructions for use of the composition and/or the lightsource, a dressing, and a device for applying and/or removing thecomposition from a treatment area.

From another aspect, there is provided a kit comprising a firstcomponent comprising a first chromophore; and a second componentcomprising a gelling agent present in an amount sufficient to gel orthicken the composition and render the biophotonic compositionsubstantially resistant to leaching such that less than 15% by weight ofthe total chromophore amount leaches out of the biophotonic compositionin use.

From a yet further aspect, there is provided a kit comprising a firstcomponent comprising a first chromophore; and a second componentcomprising a gelling agent, wherein, in combination, the first componentand the second component form a biophotonic composition substantiallyresistant to leaching such that less than 15% by weight of the totalchromophore amount leaches out of the biophotonic composition in use.The first component and/or the second component may individually also beresistant to leaching.

From another aspect, there is provided a kit comprising: a firstcomponent comprising a composition as described above, and a secondcomponent comprising an oxygen-releasing agent. Specifically, the firstcomponent may comprise a first chromophore and a gelling agent, whereinthe composition of the first component, as well as the combined firstand second component composition, are substantially resistant toleaching such that less than 15% by weight of the total chromophoreamount leaches out in use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts absorption of light in the various layers of the skin(Samson et al. Evidence Report/Technology Assessment 2004, 111, pages1-97).

FIG. 2 illustrates the Stokes' shift.

FIG. 3 illustrates the absorption and emission spectra of donor andacceptor chromophores. The spectral overlap between the absorptionspectrum of the acceptor chromophore and the emission spectrum of thedonor chromophore is also shown.

FIG. 4 is a schematic of a Jablonski diagram that illustrates thecoupled transitions involved between a donor emission and acceptorabsorbance.

FIG. 5 depicts the experimental setup of an in vitro release test forevaluating leaching of the chromophore(s) of the biophotoniccompositions (Example 6).

FIGS. 6a and 6b are absorbance and emission spectra, respectively, of acomposition according to certain embodiments of the present disclosurewhich includes Eosin and Fluorescein in a gel (Example 1).

FIGS. 7a and 7b are absorbance and emission spectra, respectively, of acomposition according to certain embodiments of the present disclosurewhich includes Eosin and Fluorescein in an aqueous solution (Example 2).

FIGS. 8a and 8b are absorbance and emission spectra, respectively, of acomposition according to certain embodiments of the present disclosurewhich includes Eosin, Fluorescein and Rose Bengal in a gel (Example 3).

FIGS. 9a and 9b are absorbance and emission spectra, respectively, of acomposition according to certain embodiments of the present disclosurewhich includes Eosin, Fluorescein and Rose Bengal in an aqueous solution(Example 4).

FIG. 10 is an emission spectrum showing the intensity over time of thelight being emitted from a biophotonic composition of the disclosuretested in Example 5.

FIG. 11 is an emission spectrum showing the intensity over time of thelight being emitted from a biophotonic composition of the disclosuretested in Example 7.

FIG. 12 shows the effect of a biophotonic composition of the disclosureon Ki67 expression (Example 10).

FIG. 13 shows that emitted fluorescence from chromophore in acomposition increases rapidly with increasing composition but slows downto a plateau with further concentration increase for Eosin Y (top) andFluorescein (bottom) (Example 13).

FIG. 14 shows that Eosin and Rose Bengal act in a synergistic manner(Example 14).

DETAILED DESCRIPTION

(1) Overview

Photodynamic therapy regimens have been developed to promote woundhealing, rejuvenate facial skins and treat various skin disorders.However, these methods require direct application of a photosensitiveagent to the target skin and/or uptake of the photosensitive agent intothe skin cells. As mentioned above, the direct contact of thephotosensitive agent with the tissue can lead to undesired side-effects,including cellular damage/destruction and systemic or localized toxicityto the patient. Moreover, many existing photodynamic therapy regimensoften demonstrate low therapeutic efficacy due to, for example, the poorupdate of the photosensitive agents into the skin cells the target site.For this reason, may regimens require a wait time of between about oneand 72 hours to allow the internalization of the photosensitizer.

Phototherapy on the other hand utilizes the therapeutic effect of light.However, expensive and sophisticated light sources are often required toprovide therapeutic wavelengths and intensities of light.

The present disclosure provides biophotonic compositions which areuseful in phototherapy and which include photoactive exogenouschromophores which can emit a therapeutic light or which can promote atherapeutic effect on a treatment site by activating other components ofthe biophotonic composition. The present disclosure also providesmethods useful for promoting wound healing, cosmetic treatment of skinsuch as skin rejuvenation, treating acne and treating other skindisorders, treating acute inflammation, which are distinguished fromconventional photodynamic therapy.

Biophotonic therapy using the present compositions does not rely oninternalization of the chromophore into cells or substantial contactwith the cells or target tissues. Therefore, the undesired side effectscaused by direct contact may be reduced, minimized, or prevented. Atmost, the chromophore has surface contact with the tissue to which thecomposition is applied, which is likely to be very short lasting due toshort treatment times. Furthermore, unlike photodynamic therapy,biophotonic therapy with embodiments of the present biophotoniccompositions does not rely on cell death or damage. In fact, Applicantshave shown in in vitro studies that a biophotonic composition accordingto an embodiment of the present disclosure reduced cell necrosis (seeExample 10).

(2) Definitions

Before continuing to describe the present disclosure in further detail,it is to be understood that this disclosure is not limited to specificcompositions or process steps, as such may vary. It must be noted that,as used in this specification and the appended claims, the singular form“a”, “an” and “the” include plural referents unless the context clearlydictates otherwise.

As used herein, the term “about” in the context of a given value orrange refers to a value or range that is within 20%, preferably within10%, and more preferably within 5% of the given value or range.

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

“Biophotonic” means the generation, manipulation, detection andapplication of photons in a biologically relevant context. In otherwords, biophotonic compositions exert their physiological effectsprimarily due to the generation and manipulation of photons.“Biophotonic composition” is a composition as described herein that maybe activated by light to produce photons for biologically relevantapplications.

“Gels” are defined as substantially dilute cross-linked systems. Gelsmay be semi-solids and exhibit substantially no flow when in the steadystate at room temperature (e.g. about 20-25° C.). By steady state ismeant herein during a treatment time and under treatment conditions.Gels, as defined herein, may be physically or chemically cross-linked.As defined herein, gels also include gel-like compositions such asviscous liquids.

“Topical” means as applied to body surfaces, such as the skin, mucousmembranes, vagina, oral cavity, internal surgical wound sites, and thelike.

Terms “chromophore”, “photoactivating agent” and “photoactivator” areused herein interchangeably. A chromophore means a chemical compound,when contacted by light irradiation, is capable of absorbing the light.The chromophore readily undergoes photoexcitation and can then transferits energy to other molecules or emit it as light.

“Photobleaching” means the photochemical destruction of a chromophore.

“Leaching” means the release of one or more components of a biophotoniccomposition (e.g., the chromophore(s) from the composition to thesurrounding environment such as for example the wound site or into thetissue being treated with the composition. The leaching properties ofthe biophotonic composition can be measured by (i) placing a 2 mm thicklayer of the biophotonic composition onto an upper side of apolycarbonate (PC) membrane having a diameter of 2.4 to 3 cm, athickness of 10 μm and a pore size of 3 μm, a lower side of the membranebeing in contact with a phosphate saline buffer solution in a receptorcompartment, (ii) allowing the biophotonic composition to rest on themembrane upper surface at room temperature and pressure for a timecorresponding to a treatment time using the biophotonic composition, and(iii) removing a sample of the solution from the receptor compartmentand measuring the concentration of the chromophore in the solution.

The term “actinic light” is intended to mean light energy emitted from aspecific light source (e.g., lamp, LED, or laser) and capable of beingabsorbed by matter (e.g. the chromophore or photoactivator definedabove). In a preferred embodiment, the actinic light is visible light.

As used herein, a “hygroscopic” substance is a substance capable oftaking up water, for example, by absorption or adsorption even atrelative humidity as low as 50%, at room temperature (e.g. about 20-25°C.).

“Impermeable membrane” means that the material contained within themembrane is sufficiently or substantially impermeable to the surroundingenvironment such that the migration of such material out of themembrane, and/or the migration of the environmental components (such aswater) into the membrane, is so low as to having substantially noadverse impact on the function or activity of the materials retainedwithin the membrane. The impermeable membrane may be ‘breathable’ inthat gas flow through the membrane is permitted whilst the flow ofliquid is not permitted. The impermeable membrane may also selectivelyallow the migration of some of the materials through the membrane butnot others.

“Wound” means an injury to any tissue, including for example, acute,subacute, delayed or difficult to heal wounds, and chronic wounds.Examples of wounds may include both open and closed wounds. Woundsinclude, for example, burns, incisions, excisions, lesions, lacerations,abrasions, puncture or penetrating wounds, gunshot wounds, surgicalwounds, contusions, hematomas, crushing injuries, ulcers (such as forexample pressure, venous, pressure or diabetic), wounds caused byperiodontitis (inflammation of the periodontium).

“Skin rejuvenation” means a process of reducing, diminishing, retardingor reversing one or more signs of skin aging. For instance, common signsof skin aging include, but are not limited to, appearance of fine linesor wrinkles, thin and transparent skin, loss of underlying fat (leadingto hollowed cheeks and eye sockets as well as noticeable loss offirmness on the hands and neck), bone loss (such that bones shrink awayfrom the skin due to bone loss, which causes sagging skin), dry skin(which might itch), inability to sweat sufficiently to cool the skin,unwanted facial hair, freckles, age spots, spider veins, rough andleathery skin, fine wrinkles that disappear when stretched, loose skin,or a blotchy complexion. According to the present disclosure, one ormore of the above signs of aging may be reduced, diminished, retarded oreven reversed by the compositions and methods of the present disclosure.

(3) Biophotonic Topical Compositions

The present disclosure provides biophotonic compositions. Biophotoniccompositions are compositions that are, in a broad sense, activated bylight (e.g., photons) of specific wavelength. These compositions containat least one exogenous chromophore which is activated by light andaccelerates the dispersion of light energy, which leads to lightcarrying on a therapeutic effect on its own, and/or to the photochemicalactivation of other agents contained in the composition (e.g.,acceleration in the breakdown process of peroxide (an oxygen-releasingagent) when such compound is present in the composition or at thetreatment site, leading to the formation of oxygen radicals, such assinglet oxygen). The composition may comprise an oxygen-releasing agentwhich, when mixed with the first chromophore and subsequently activatedby light, can be photochemically activated which may lead to theformation of oxygen radicals, such as singlet oxygen.

In some aspects, the present disclosure provides biophotoniccompositions comprising at least a first chromophore in a medium,wherein the composition is substantially resistant to leaching such thata low or negligible chromophore amount leaches out of the biophotoniccomposition into a treatment site (e.g. tissue) onto which thecomposition is applied during treatment. In certain embodiments, this isachieved by the medium comprising a gelling agent which slows orrestricts movement or leaching of the chromophore. In other embodiments,this is achieved by provision of an encapsulating membrane around thefirst chromophore in the medium. In this way, contact of the chromophoreand the tissue can be minimized or avoided.

In some aspects, biophotonic compositions of the present disclosure donot stain the tissue onto which they are topically applied duringtreatment. Staining is determined by visually assessing whether thebiophotonic composition colorizes white test paper saturated with 70% byvolume ethanol/30% by volume water solution placed in contact with thebiophotonic composition for a period of time corresponding to a desiredtreatment time. In some embodiments, a biophotonic composition of thepresent disclosure does not visually colorize white test paper saturatedwith a 70% by volume ethanol/30% by volume water solution placed incontact with the biophotonic composition under atmospheric pressure fora time corresponding to a desired treatment time. In certainembodiments, the time corresponding to a treatment time is at leastabout 5 minutes, at least about 10 minutes, 15 minutes, 20 minutes, 25minutes or 30 minutes.

When a chromophore absorbs a photon of a certain wavelength, it becomesexcited. This is an unstable condition and the molecule tries to returnto the ground state, giving away the excess energy. For somechromophores, it is favorable to emit the excess energy as light whentransforming back to the ground state. This process is calledfluorescence. The peak wavelength of the emitted fluorescence is shiftedtowards longer wavelengths compared to the absorption wavelengths(‘Stokes' shift’). The emitted fluorescent energy can then betransferred to the other components of the composition or to a treatmentsite on to which the biophotonic composition is topically applied. FIG.1 illustrates the different penetrative depths of different wavelengthof light. Differing wavelengths of light may have different andcomplementary therapeutic effects on tissue. Stokes' shift isillustrated in FIG. 2.

Without being bound to theory, it is thought that fluorescent lightemitted by photoactivated chromophores may have therapeutic propertiesdue to its femto-, pico- or nano-second emission properties which may berecognized by biological cells and tissues, leading to favorablebiomodulation. Furthermore, the emitted fluorescent light has a longerwavelength and hence a deeper penetration into the tissue than theactivating light. Irradiating tissue with such a broad range ofwavelengths, including in some embodiments the activating light whichpasses through the composition, may have different and complementaryeffects on the cells and tissues. Moreover, in embodiments of thecomposition containing oxygen-releasing agent(s), micro-bubbling withinthe composition has been observed by the inventor which may beassociated with the generation of oxygen species by the photoactivatedchromophores. This may have a physical impact on the tissue to which itis applied, for example by dislodging biofilm and debridement ofnecrotic tissue or providing a pressure stimulation. The biofilm canalso be pre-treated with an oxygen-releasing agent to weaken the biofilmbefore treating with the composition of the present disclosure.

Certain embodiments of the biophotonic compositions of the presentdisclosure are substantially transparent/translucent and/or have highlight transmittance in order to permit light dissipation into andthrough the composition. In this way, the area of tissue under thecomposition can be treated both with the fluorescent light emitted bythe composition and the light irradiating the composition to activateit, which may benefit from the different therapeutic effects of lighthaving different wavelengths.

The % transmittance of the biophotonic composition can be measured inthe range of wavelengths from 250 nm to 800 nm using, for example, aPerkin-Elmer Lambda 9500 series UV-visible spectrophotometer.Alternatively, a Synergy HT spectrophotometer (BioTek Instrument, Inc.)can be used in the range of wavelengths from 380 nm to 900 nm.

Transmittance is calculated according to the following equation:

$A_{\lambda} = {{\log_{10}\frac{I_{0}}{I}} = {\log_{10}{\frac{1}{T}.}}}$where A is absorbance, T is transmittance, I₀ is intensity of radiationbefore passing through material, I is intensity of light passing throughmaterial.The values can be normalized for thickness. As stated herein, %transmittance (translucency) is as measured for a 2 mm thick sample at awavelength of 526 nm. It will be clear that other wavelengths can beused.

In some embodiments, the biophotonic composition has a transparency ortranslucency that exceeds 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%,75%, 80%, or 85%. In some embodiments, the transparency exceeds 70%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.All transmittance values reported herein are as measured on a 2 mm thicksample using the Synergy HT spectrophotometer at a wavelength of 526 nm.

Embodiments of the biophotonic compositions of the present disclosureare for topical uses. The biophotonic composition can be in the form ofa semi-solid or viscous liquid, having properties which limit leachingof the chromophore(s) from the composition to less than 15% by weight.Preferably, the biophotonic compositions are a gel or are gel-like,including viscous liquids, and which have a spreadable consistency atroom temperature (e.g. about 20-25° C.), prior to illumination. Byspreadable is meant that the composition can be topically applied to atreatment site at a thickness of about 2 mm. Spreadable compositions canconform to a topography of a treatment site, e.g. a wound. This can haveadvantages over a non-conforming material in that a better and/or morecomplete illumination of the treatment site can be achieved.

These compositions may be described based on the components making upthe composition. Additionally or alternatively, the compositions of thepresent disclosure have functional and structural properties and theseproperties may also be used to define and describe the compositions.Individual components of the composition of the present disclosure aredetailed as below.

(a) Chromophores

The biophotonic topical compositions of the present disclosure compriseone or more chromophores, which can be considered exogenous, e.g., arenot naturally present in skin or tissue. The chromophores are containedor held within the biophotonic composition such that they do notsubstantially contact the target tissue to which the composition isapplied during a treatment time. In this way, the beneficial andtherapeutic properties of the chromophore can be harnessed without thepossibly damaging effects caused by chromophore-cell contact.

Suitable chromophores can be fluorescent dyes (or stains), althoughother dye groups or dyes (biological and histological dyes, foodcolorings, carotenoids, naturally occurring fluorescent and other dyes)can also be used. Suitable chromophores can be those that are GenerallyRegarded As Safe (GRAS), although chromophores which are not welltolerated by the skin or other tissues can be included in thebiophotonic composition as contact with the skin is minimal in use dueto the leaching—barrier nature of the composition.

In certain embodiments, the biophotonic topical composition of thepresent disclosure comprises a first chromophore which undergoes partialor complete photobleaching upon application of light. By photobleachingis meant a photochemical destruction of the chromophore which cangenerally be visualized as a loss of color.

In some embodiments, the first chromophore absorbs at a wavelength inthe range of the visible spectrum, such as at a wavelength of about380-800 nm, 380-700, or 380-600 nm. In other embodiments, the firstchromophore absorbs at a wavelength of about 200-800 nm, 200-700 nm,200-600 nm or 200-500 nm. In one embodiment, the first chromophoreabsorbs at a wavelength of about 200-600 nm. In some embodiments, thefirst chromophore absorbs light at a wavelength of about 200-300 nm,250-350 nm, 300-400 nm, 350-450 nm, 400-500 nm, 400-600 nm, 450-650 nm,600-700 nm, 650-750 nm or 700-800 nm.

It will be appreciated to those skilled in the art that opticalproperties of a particular chromophore may vary depending on thechromophore's surrounding medium. Therefore, as used herein, aparticular chromophore's absorption and/or emission wavelength (orspectrum) corresponds to the wavelengths (or spectrum) measured in abiophotonic composition of the present disclosure.

The biophotonic compositions disclosed herein may include at least oneadditional chromophore. Combining chromophores may increasephoto-absorption by the combined dye molecules and enhance absorptionand photo-biomodulation selectivity. This creates multiple possibilitiesof generating new photosensitive, and/or selective chromophoresmixtures.

When such multi-chromophore compositions are illuminated with light,energy transfer can occur between the chromophores. This process, knownas resonance energy transfer, is a photophysical process through whichan excited ‘donor’ chromophore (also referred to herein as firstchromophore) transfers its excitation energy to an ‘acceptor’chromophore (also referred to herein as second chromophore). Theefficiency and directedness of resonance energy transfer depends on thespectral features of donor and acceptor chromophores. In particular, theflow of energy between chromophores is dependent on a spectral overlapreflecting the relative positioning and shapes of the absorption andemission spectra. For energy transfer to occur the emission spectrum ofthe donor chromophore overlap with the absorption spectrum of theacceptor chromophore (FIG. 3).

Energy transfer manifests itself through decrease or quenching of thedonor emission and a reduction of excited state lifetime accompaniedalso by an increase in acceptor emission intensity. FIG. 4 is aJablonski diagram that illustrates the coupled transitions involvedbetween a donor emission and acceptor absorbance.

To enhance the energy transfer efficiency, the donor chromophore shouldhave good abilities to absorb photons and emit photons. Furthermore, itis thought that the more overlap there is between the donorchromospheres' emission spectra and the acceptor chromophore'sabsorption spectra, the better a donor chromophore can transfer energyto the acceptor chromophore.

In certain embodiments, the biophotonic topical composition of thepresent disclosure further comprises a second chromophore. In someembodiments, the first chromophore has an emission spectrum thatoverlaps at least about 80%, 50%, 40%, 30%, 20%, 10% with an absorptionspectrum of the second chromophore. In one embodiment, the firstchromophore has an emission spectrum that overlaps at least about 20%with an absorption spectrum of the second chromophore. In someembodiments, the first chromophore has an emission spectrum thatoverlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%,35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of thesecond chromophore.

% spectral overlap, as used herein, means the % overlap of a donorchromophore's emission wavelength range with an acceptor chromophore'sabsorption wavelength rage, measured at spectral full width quartermaximum (FWQM). For example, FIG. 3 shows the normalized absorption andemission spectra of donor and acceptor chromophores. The spectral FWQMof the acceptor chromophore's absorption spectrum is from about 60 nm(515 nm to about 575 nm). The overlap of the donor chromophore'sspectrum with the absorption spectrum of the acceptor chromophore isabout 40 nm (from 515 nm to about 555 nm). Thus, the % overlap can becalculated as 40 nm/60 nm×100=66.6%.

In some embodiments, the second chromophore absorbs at a wavelength inthe range of the visible spectrum. In certain embodiments, the secondchromophore has an absorption wavelength that is relatively longer thanthat of the first chromophore within the range of about 50-250, 25-150or 10-100 nm.

As discussed above, the application of light to the compositions of thepresent disclosure can result in a cascade of energy transfer betweenthe chromophores. In certain embodiments, such a cascade of energytransfer provides photons that penetrate the epidermis, dermis and/ormucosa at the target tissue, including, such as, a site of wound, or atissue afflicted with acne or a skin disorder. In some embodiments, sucha cascade of energy transfer is not accompanied by concomitantgeneration of heat. In some other embodiments, the cascade of energytransfer does not result in tissue damage.

Optionally, when the biophotonic topical composition comprises a firstand a second chromophore, the first chromophore is present in an amountof about 0.005-40% per weight of the composition, and the secondchromophore is present in an amount of about 0.001-40% per weight of thecomposition. In certain embodiments, the total weight per weight ofchromophore or combination of chromophores may be in the amount of about0.005-40.001% per weight of the composition. In certain embodiments, thefirst chromophore is present in an amount of about 0.005-1%, 0.01-2%,0.02-1%, 0.02-2%, 0.05-1%, 0.05-2%, 0.05-1%, 0.05-2%, 1-5%, 2.5-7.5%,5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%,22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weightof the composition. In certain embodiments, the second chromophore ispresent in an amount of about 0.001-1%, 0.001-2%, 0.001-0.01%,0.01-0.1%, 0.1-1.0%, 1-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%,12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%,30-35%, 32.5-37.5%, or 35-40% per weight of the composition. In certainembodiments, the total weight per weight of chromophore or combinationof chromophores may be in the amount of about 0.005-1%, 0.01-2%,0.05-2%, 0.5-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%,12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%,30-35%, 32.5-37.5%, or 35-40.05% per weight of the composition.

In some embodiments, the chromophore or chromophores are selected suchthat their emitted fluorescent light, on photoactivation, is within oneor more of the green, yellow, orange, red and infrared portions of theelectromagnetic spectrum, for example having a peak wavelength withinthe range of about 490 nm to about 800 nm. In certain embodiments, theemitted fluorescent light has a power density of between 0.005 to about10 mW/cm², about 0.5 to about 5 mW/cm².

Suitable chromophores that may be used in the biophotonic topicalcompositions of the present disclosure include, but are not limited tothe following:

Chlorophyll Dyes

Exemplary chlorophyll dyes include but are not limited to chlorophyll a;chlorophyll b; oil soluble chlorophyll; bacteriochlorophyll a;bacteriochlorophyll b; bacteriochlorophyll c; bacteriochlorophyll d;protochlorophyll; protochlorophyll a; amphiphilic chlorophyll derivative1; and amphiphilic chlorophyll derivative 2.

Xanthene Derivatives

Exemplary xanthene dyes include but are not limited to Eosin B(4′,5′-dibromo,2′,7′-dinitr-o-fluorescein, dianion); eosin Y; eosin Y(2′,4′,5′,7′-tetrabromo-fluoresc-ein, dianion); eosin(2′,4′,5′,7′-tetrabromo-fluorescein, dianion); eosin(2′,4′,5′,7′-tetrabromo-fluorescein, dianion) methyl ester; eosin(2′,4′,5′,7′-tetrabromo-fluorescein, monoanion) p-isopropylbenzyl ester;eosin derivative (2′,7′-dibromo-fluorescein, dianion); eosin derivative(4′,5′-dibromo-fluorescein, dianion); eosin derivative(2′,7′-dichloro-fluorescein, dianion); eosin derivative(4′,5′-dichloro-fluorescein, dianion); eosin derivative(27-diiodo-fluorescein, dianion); eosin derivative(4′,5′-diiodo-fluorescein, dianion); eosin derivative(tribromo-fluorescein, dianion); eosin derivative(2′,4′,5′,7′-tetrachlor-o-fluorescein, dianion); eosin; eosindicetylpyridinium chloride ion pair; erythrosin B(2′,4′,5′,7′-tetraiodo-fluorescein, dianion); erythrosin; erythrosindianion; erythiosin B; fluorescein; fluorescein dianion; phloxin B(2′,4′,5′,7′-tetrabromo-3,4,5,6-tetrachloro-fluorescein, dianion);phloxin B (tetrachloro-tetrabromo-fluorescein); phloxine B; rose bengal(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, dianion); pyroninG, pyronin J, pyronin Y; Rhodamine dyes such as rhodamines include4,5-dibromo-rhodamine methyl ester; 4,5-dibromo-rhodamine n-butyl ester;rhodamine 101 methyl ester; rhodamine 123; rhodamine 6G; rhodamine 6Ghexyl ester; tetrabromo-rhodamine 123; and tetramethyl-rhodamine ethylester.

Methylene Blue Dyes

Exemplary methylene blue derivatives include but are not limited to1-methyl methylene blue; 1,9-dimethyl methylene blue; methylene blue;methylene blue (16 .mu.M); methylene blue (14 .mu.M); methylene violet;bromomethylene violet; 4-iodomethylene violet;1,9-dimethyl-3-dimethyl-amino-7-diethyl-a-mino-phenothiazine; and1,9-dimethyl-3-diethylamino-7-dibutyl-amino-phenot-hiazine.

Azo Dyes

Exemplary azo (or diazo-) dyes include but are not limited to methylviolet, neutral red, para red (pigment red 1), amaranth (Azorubine S),Carmoisine (azorubine, food red 3, acid red 14), allura red AC (FD&C40), tartrazine (FD&C Yellow 5), orange G (acid orange 10), Ponceau 4R(food red 7), methyl red (acid red 2), and murexide-ammonium purpurate.

In some aspects of the disclosure, the one or more chromophores of thebiophotonic composition disclosed herein can be independently selectedfrom any of Acid black 1, Acid blue 22, Acid blue 93, Acid fuchsin, Acidgreen, Acid green 1, Acid green 5, Acid magenta, Acid orange 10, Acidred 26, Acid red 29, Acid red 44, Acid red 51, Acid red 66, Acid red 87,Acid red 91, Acid red 92, Acid red 94, Acid red 101, Acid red 103, Acidroseine, Acid rubin, Acid violet 19, Acid yellow 1, Acid yellow 9, Acidyellow 23, Acid yellow 24, Acid yellow 36, Acid yellow 73, Acid yellowS, Acridine orange, Acriflavine, Alcian blue, Alcian yellow, Alcoholsoluble eosin, Alizarin, Alizarin blue 2RC, Alizarin carmine, Alizarincyanin BBS, Alizarol cyanin R, Alizarin red S, Alizarin purpurin,Aluminon, Amido black 10B, Amidoschwarz, Aniline blue WS, Anthraceneblue SWR, Auramine O, Azocannine B, Azocarmine G, Azoic diazo 5, Azoicdiazo 48, Azure A, Azure B, Azure C, Basic blue 8, Basic blue 9, Basicblue 12, Basic blue 15, Basic blue 17, Basic blue 20, Basic blue 26,Basic brown 1, Basic fuchsin, Basic green 4, Basic orange 14, Basic red2 (Saffranin O), Basic red 5, Basic red 9, Basic violet 2, Basic violet3, Basic violet 4, Basic violet 10, Basic violet 14, Basic yellow 1,Basic yellow 2, Biebrich scarlet, Bismarck brown Y, Brilliant crystalscarlet 6R, Calcium red, Carmine, Carminic acid (acid red 4), Celestineblue B, China blue, Cochineal, Coelestine blue, Chrome violet CG,Chromotrope 2R, Chromoxane cyanin R, Congo corinth, Congo red, Cottonblue, Cotton red, Croceine scarlet, Crocin, Crystal ponceau 6R, Crystalviolet, Dahlia, Diamond green B, DiOC6, Direct blue 14, Direct blue 58,Direct red, Direct red 10, Direct red 28, Direct red 80, Direct yellow7, Eosin B, Eosin Bluish, Eosin, Eosin Y, Eosin yellowish, Eosinol, Eriegarnet B, Eriochrome cyanin R, Erythrosin B, Ethyl eosin, Ethyl green,Ethyl violet, Evans blue, Fast blue B, Fast green FCF, Fast red B, Fastyellow, Fluorescein, Food green 3, Gallein, Gallamine blue, Gallocyanin,Gentian violet, Haematein, Haematine, Haematoxylin, Helio fast rubinBBL, Helvetia blue, Hematein, Hematine, Hematoxylin, Hoffman's violet,Imperial red, Indocyanin green, Ingrain blue, Ingrain blue 1, Ingrainyellow 1, INT, Kermes, Kermesic acid, Kernechtrot, Lac, Laccaic acid,Lauth's violet, Light green, Lissamine green SF, Luxol fast blue,Magenta 0, Magenta I, Magenta II, Magenta III, Malachite green,Manchester brown, Martius yellow, Merbromin, Mercurochrome, Metanilyellow, Methylene azure A, Methylene azure B, Methylene azure C,Methylene blue, Methyl blue, Methyl green, Methyl violet, Methyl violet2B, Methyl violet 10B, Mordant blue 3, Mordant blue 10, Mordant blue 14,Mordant blue 23, Mordant blue 32, Mordant blue 45, Mordant red 3,Mordant red 11, Mordant violet 25, Mordant violet 39 Naphthol blueblack, Naphthol green B, Naphthol yellow S, Natural black 1, Naturalred, Natural red 3, Natural red 4, Natural red 8, Natural red 16,Natural red 25, Natural red 28, Natural yellow 6, NBT, Neutral red, Newfuchsin, Niagara blue 3B, Night blue, Nile blue, Nile blue A, Nile blueoxazone, Nile blue sulphate, Nile red, Nitro BT, Nitro blue tetrazolium,Nuclear fast red, Oil red O, Orange G, Orcein, Pararosanilin, PhloxineB, phycobilins, Phycocyanins, Phycoerythrins. Phycoerythrincyanin (PEC),Phthalocyanines, Picric acid, Ponceau 2R, Ponceau 6R, Ponceau B, Ponceaude Xylidine, Ponceau S, Primula, Purpurin, Pyronin B, Pyronin G, PyroninY, Rhodamine B, Rosanilin, Rose bengal, Saffron, Safranin O, Scarlet R,Scarlet red, Scharlach R, Shellac, Sirius red F3B, Solochrome cyanin R,Soluble blue, Solvent black 3, Solvent blue 38, Solvent red 23, Solventred 24, Solvent red 27, Solvent red 45, Solvent yellow 94, Spiritsoluble eosin, Sudan III, Sudan IV, Sudan black B, Sulfur yellow S,Swiss blue, Tartrazine, Thioflavine S, Thioflavine T, Thionin, Toluidineblue, Toluyline red, Tropaeolin G, Trypaflavine, Trypan blue, Uranin,Victoria blue 4R, Victoria blue B, Victoria green B, Water blue I, Watersoluble eosin, Xylidine ponceau, or Yellowish eosin.

In certain embodiments, the composition of the present disclosureincludes any of the chromophores listed above, or a combination thereof,so as to provide a biophotonic impact at the application site. This is adistinct application of these agents and differs from the use ofchromophores as simple stains or as a catalyst for photo-polymerization.

Chromophores can be selected, for example, on their emission wavelengthproperties in the case of fluorophores, on the basis of their energytransfer potential, their ability to generate reactive oxygen species,or their antimicrobial effect. These needs may vary depending on thecondition requiring treatment. For example, chlorophylls may have anantimicrobial effect on bacteria found on the face.

In some embodiments, the composition includes Eosin Y as a firstchromophore and any one or more of Rose Bengal, Erythrosin, Phloxine Bas a second chromophore. It is believed that these combinations have asynergistic effect as Eosin Y can transfer energy to Rose Bengal,Erythrosin or Phloxine B when activated. This transferred energy is thenemitted as fluorescence or by production of reactive oxygen species.This absorbed and re-emitted light is thought to be transmittedthroughout the composition, and also to be transmitted into the site oftreatment.

In further embodiments, the composition includes the followingsynergistic combinations: Eosin Y and Fluorescein; Fluorescein and RoseBengal; Erythrosine in combination with Eosin Y, Rose Bengal orFluorescein; Phloxine B in combination with one or more of Eosin Y, RoseBengal, Fluorescein and Erythrosine. Other synergistic chromophorecombinations are also possible.

By means of synergistic effects of the chromophore combinations in thecomposition, chromophores which cannot normally be activated by anactivating light (such as a blue light from an LED) can be activatedthrough energy transfer from chromophores which are activated by theactivating light. In this way, the different properties ofphotoactivated chromophores can be harnessed and tailored according tothe cosmetic or the medical therapy required.

For example, Rose Bengal can generate a high yield of singlet oxygenwhen photoactivated in the presence of molecular oxygen, however it hasa low quantum yield in terms of emitted fluorescent light. Rose Bengalhas a peak absorption around 540 nm and so is normally activated bygreen light. Eosin Y has a high quantum yield and can be activated byblue light. By combining Rose Bengal with Eosin Y, one obtains acomposition which can emit therapeutic fluorescent light and generatesinglet oxygen when activated by blue light. In this case, the bluelight photoactivates Eosin Y which transfers some of its energy to RoseBengal as well as emitting some energy as fluorescence.

Chromophore combinations can also have a synergistic effect in terms oftheir photoactivated state. For example, two chromophores may be used,one of which emits fluorescent light when activated in the blue andgreen range, and the other which emits fluorescent light in the red,orange and yellow range, thereby complementing each other andirradiating the target tissue with a broad wavelength of light havingdifferent depths of penetration into target tissue and differenttherapeutic effects.

(b) Gelling Agent

The present disclosure provides biophotonic compositions that compriseat least a first chromophore and a gelling agent, wherein the gellingagent provides a barrier such that the chromophore(s) of the biophotonictopical compositions are substantially not in contact with the targettissue. The gelling agent, when present in the biophotonic compositionsof the present disclosure, can render the compositions substantiallyresistant to leaching such that the chromophore(s) or photosensitiveagent(s) of the biophotonic topical compositions are not in substantialcontact with the target tissue.

In certain embodiments, the biophotonic topical composition allows lessthan 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or 0.1%, or essentiallynone of said chromophore content to leach out of the biophotoniccomposition.

In some embodiments, the biophotonic composition limits leaching of thefirst chromophore such that less than 15% of total chromophore amountcan leach out into tissue during a treatment time in which thecomposition is topically applied onto tissue and illuminated with light.In some embodiments, the biophotonic composition limits leaching of thefirst chromophore such that less than 30%, 25%, 20%, 15%, 10%, 5%, 1%,0.8%, 0.5% or 0.1% or essentially 0% of total chromophore amount canleach out into tissue during a treatment time in which the compositionis topically applied onto tissue and illuminated with light. In someembodiments, the treatment time is at least about 5 minutes, at leastabout 10 minutes, at least about 15 minutes, at least about 20 minutes,at least about 25 minutes or at least about 30 minutes.

Leaching can be determined as described in Example 6 (see FIG. 5). Insome embodiments, a biophotonic composition of the present disclosureallows less than 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or 0.1% oressentially 0% of the total chromophore amount to leach out of thebiophotonic composition as through a porous membrane into an aqueoussolution when the biophotonic composition is placed in contact with theaqueous solution through the porous membrane for a time corresponding toa desired treatment time. In certain embodiments, the time correspondingto a treatment time is at least about 5 minutes, at least about 10minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes.

A gelling agent for use according to the present disclosure may compriseany ingredient suitable for use in a topical biophotonic formulation asdescribed herein. The gelling agent may be an agent capable of forming across-linked matrix, including physical and/or chemical cross-links. Thegelling agent is preferably biocompatible, and may be biodegradable. Insome embodiments, the gelling agent is able to form a hydrogel or ahydrocolloid. An appropriate gelling agent is one that can form aviscous liquid or a semisolid. In preferred embodiments, the gellingagent and/or the composition has appropriate light transmissionproperties. It is also important to select a gelling agent which willallow biophotonic activity of the chromophore(s). For example, somechromophores require a hydrated environment in order to fluoresce. Thegelling agent may be able to form a gel by itself or in combination withother ingredients such as water or another gelling agent, or whenapplied to a treatment site, or when illuminated with light.

The gelling agent according to various embodiments of the presentdisclosure may include, but not be limited to, polyalkylene oxides,particularly polyethylene glycol and poly(ethylene oxide)-poly(propyleneoxide) copolymers, including block and random copolymers; polyols suchas glycerol, polyglycerol (particularly highly branched polyglycerol),propylene glycol and trimethylene glycol substituted with one or morepolyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated glycerol,mono- and di-polyoxy-ethylated propylene glycol, and mono- anddi-polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,polyoxyethylated glucose; acrylic acid polymers and analogs andcopolymers thereof, such as polyacrylic acid per se, polymethacrylicacid, poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxideacrylate) and copolymers of any of the foregoing, and/or with additionalacrylate species such as aminoethyl acrylate and mono-2-(acryloxy)-ethylsuccinate; polymaleic acid; poly(acrylamides) such as polyacrylamide perse, poly(methacrylamide), poly(dimethylacrylamide), andpoly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as poly(vinylalcohol); poly(N-vinyl lactams) such as poly(vinyl pyrrolidone),poly(N-vinyl caprolactam), and copolymers thereof, polyoxazolines,including poly(methyloxazoline) and poly(ethyloxazoline); andpolyvinylamines.

The gelling agent according to certain embodiments of the presentdisclosure may include a polymer selected from any of synthetic orsemi-synthetic polymeric materials, polyacrylate copolymers, cellulosederivatives and polymethyl vinyl ether/maleic anhydride copolymers. Insome embodiments, the hydrophilic polymer comprises a polymer that is ahigh molecular weight (i.e., molar masses of more than about 5,000, andin some instances, more than about 10,000, or 100,000, or 1,000,000)and/or cross-linked polyacrylic acid polymer. In some embodiments, thepolymer is a polyacrylic acid polymer and has a viscosity in the rangeof about 10,000-100,000; 10,000-80,000; 15,000-80,000; 10,000-70,000;15,000-70,000; 10,000-60,000; 10,000-50,000; 10,000-40,000;20,000-100,000; 25,000-90,000; 30,000-80,000; 30,000-70,000;30,000-60,000; 25,000-40,000 cP. In certain embodiment, the polymer is ahigh molecular weight, and/or cross-linked polyacrylic acid polymer,where the polyacrylic acid polymer has a viscosity in the range of about10,000-80,000 cP.

In some embodiments, the gelling agent comprises a carbomer. Carbomersare synthetic high molecular weight polymer of acrylic acid that arecross-linked with either allylsucrose or allylethers of pentaerythritolhaving a molecular weight of about 3×10⁶. The gelation mechanism dependson neutralization of the carboxylic acid moiety to form a soluble salt.The polymer is hydrophilic and produces sparkling clear gels whenneutralized. Carbomer gels possess good thermal stability in that gelviscosity and yield value are essentially unaffected by temperature. Asa topical product, carbomer gels possess optimum rheological properties.The inherent pseudoplastic flow permits immediate recovery of viscositywhen shear is terminated and the high yield value and quick break makeit ideal for dispensing. Aqueous solution of Carbopol® is acidic innature due to the presence of free carboxylic acid residues.Neutralization of this solution cross-links and gelatinizes the polymerto form a viscous integral structure of desired viscosity.

Carbomers are available as fine white powders which disperse in water toform acidic colloidal suspensions (a 1% dispersion has approx. pH 3) oflow viscosity. Neutralization of these suspensions using a base, forexample sodium, potassium or ammonium hydroxides, low molecular weightamines and alkanolamines, results in the formation of translucent gels.Nicotine salts such as nicotine chloride form stable water-solublecomplexes with carbomers at about pH 3.5 and are stabilized at anoptimal pH of about 5.6.

In some embodiments of the disclosure, the carbomer is Carbopol. Suchpolymers are commercially available from B.F. Goodrich or Lubrizol underthe designation Carbopol® 71 G NF, 420, 430, 475, 488, 493, 910, 934,934P, 940, 971PNF, 974P NF, 980 NF, 981 NF and the like. Carbopols areversatile controlled-release polymers, as described by Brock(Pharmacotherapy, 14:430-7 (1994)) and Durrani (Pharmaceutical Res.(Supp.) 8:S-135 (1991)), and belong to a family of carbomers which aresynthetic, high molecular weight, non-linear polymers of acrylic acid,crosslinked with polyalkenyl polyether. In some embodiments, thecarbomer is Carbopol® 974P NF, 980 NF, 5984 EP, ETD 2020NF, Ultrez 10NF, 934 NF, 934P NF or 940 NF. In certain embodiments, the carbomer isCarbopol® 980 NF, ETD 2020 NF, Ultrez 10 NF, Ultrez 21 or 1382 Polymer,1342 NF, 940 NF. For example, 0.05 to 10%, preferably 0.5 to 5%, morepreferably 1 to 3% by weight of the final composition of a highmolecular weight carbopol can be present as the gelling agent and whichcan form a gel having a viscosity of more than about 10,000 cP, orpreferably more than about 15,000 cP.

In certain embodiments, the gelling agent comprises a hygroscopic and/ora hydrophilic material which may be used for their water attractingproperties, which may also prevent or limit leaching of the chromophore.The hygroscopic or hydrophilic material may include, but is not limitedto, glucosamine, polysaccharides, glycosaminoglycan, poly(vinylalcohol), poly(2-hydroxyethylmethylacrylate), polyethylene oxide,collagen, chitosan, alginate, a poly(acrylonitrile)-based hydrogel,poly(ethylene glycol)/poly(acrylic acid) interpenetrating polymernetwork hydrogel, polyethylene oxide-polybutylene terephthalate,hyaluronic acid, high-molecular-weight polyacrylic acid, poly(hydroxyethylmethacrylate), poly(ethylene glycol), tetraethylene glycoldiacrylate, polyethylene glycol methacrylate, and poly(methylacrylate-co-hydroxyethyl acrylate).

The one or more gelling agents can be selected according to theirability to prevent chromophore leaching. For example, gelling agentswhich increase the viscosity of the biophotonic composition can beselected. In some embodiments, the viscosity of the biophotoniccomposition is 15,000-100,000, 15,000-90,000, 15,000-80,000,20,000-80,000, 20,000-70,000, 20,000-50,000, 10,000-50,000,15,000-50,000, 10,000-40,000, 15,000-40,000 cP. A composition withsufficiently high viscosity parameters can prevent or limit the leachingof chromophores from the composition. Viscosity of the biophotoniccompositions of the present disclosure is as measured using a cone/plateviscometer (Wells-Brookfield) using a CP -51 and measuring viscosity ata speed of 2 rpm and making sure that the torque is >10%. Spindle mustrotate at least 5 times before a viscosity reading is taken.Alternatively a Brookfield DV-II+Pro viscometer with Spindle 7, 50 rpm,1 minute can be used.

Gelling agents which include lipids or other coating agents which cancoat the chromophores can also be used to limit or prevent leaching. Thegelling agent may be protein-based/naturally derived material such assodium hyaluronate, gelatin or collagen, or the like. The gelling agentmay be a polysaccharide such as starch, chitosan, chitin, agarose, agar,locust bean gum, carrageenan, gellan gum, pectin, alginate, xanthan,guar gum, and the like.

In one embodiment, the composition can include up to about 2% by weightof the final composition of sodium hyaluronate as the single gellingagent. In another embodiment, the composition can include more thanabout 4%, preferably more than about 5%, by weight of the finalcomposition of gelatin as the single gelling agent. In anotherembodiment, the composition can include up to about 10%, preferably upto about 8%, starch as the single gelling agent. In yet anotherembodiment, the composition can include more than about 5%, preferablymore than about 10%, by weight of the final composition of collagen asthe gelling agent. In further embodiments, about 0.1-10%, or about0.5-3%, by weight of the final composition of chitin can be used as thegelling agent. In other embodiments, 0.5%-5% by weight of the finalcomposition of corn starch, or 5-10% by weight of the final compositionof starch can be used as the gelling agent. In certain otherembodiments, more than 2.5 wt % by weight of the final composition ofalginate can be used in the composition as the gelling agent. In otherembodiments, the percentages by weight percent of the final compositionof the gelling agents can be as follows: cellulose gel (about 0.3-2.0%),konjac gum (0.5-0.7%), carrageenan gum (0.02-2.0%), xanthan gum(0.01-2.0%), acacia gum (3-30%), agar (0.04-1.2%), guar gum (0.1-1%),locust bean gum (0.15-0.75%), pectin (0.1-0.6%), tara gum (0.1-1.0%),polyvinylypyrrolidone (1-5%), sodium polyacrylate (1-10%). Other gellingagents can be used in amounts sufficient to gel the composition or tosufficiently thicken the composition to avoid or minimize leaching ofthe chromophore(s). It will be appreciated that lower amounts of theabove gelling agents may be used in the presence of another gellingagent or a thickener.

The biophotonic composition of the present disclosure may be furtherencapsulated, e.g., in a membrane. Such a membrane may be transparent,and/or substantially, or fully impermeable. The membrane may beimpermeable to liquid but permeable to gases such as air. In certainembodiments, the composition may form a membrane that encapsulates thechromophore(s) of the biophotonic topical composition, where themembrane may be substantially impermeable to liquid and/or gas.

In certain embodiments, the retention of the chromophore in thecomposition during the treatment time can be achieved by providing amembrane around a chromophore(s) in a carrier medium. In this case, itis the membrane which limits or stops leaching of the chromophore suchas by providing a barrier. The carrier medium can be a liquidencapsulated by the membrane, wherein the membrane is sufficientlyresistant to chromophore leaching such that less than 15% of the totalchromophore amount leaches out of the encapsulated composition. Themembrane may be formed of one or more lipidic agents, polymers, gelatin,cellulose or cyclodextrins, or the like. Preferably, the membrane istranslucent or transparent to allow light to infiltrate to and from thechromophore(s). In one embodiment, the composition is a dendrimer withan outer membrane comprising poly(propylene amine). In anotherembodiment, the outer membrane comprises gelatin.

(c) Oxygen-Releasing Agents

According to certain embodiments, the compositions of the presentdisclosure may optionally further comprise one or more additionalcomponents, such as oxygen-releasing agents. For instance, thebiophotonic topical composition of the present disclosure may optionallycomprise oxygen-releasing agents as a source of oxygen. Peroxidecompounds are oxygen-releasing agents that contain the peroxy group(R—O—O—R), which is a chainlike structure containing two oxygen atoms,each of which is bonded to the other and a radical or some element.

When a biophotonic composition of the present disclosure comprising anoxygen-releasing agent is illuminated with light, the chromophore(s) areexcited to a higher energy state. When the chromophore(s)' electronsreturn to a lower energy state, they emit photons with a lower energylevel, thus causing the emission of light of a longer wavelength(Stokes' shift). In the proper environment, some of this energy releaseis transferred to oxygen or the reactive hydrogen peroxide and causesthe formation of oxygen radicals, such as singlet oxygen. The singletoxygen and other reactive oxygen species generated by the activation ofthe biophotonic composition are thought to operate in a hormeticfashion. That is, a health beneficial effect that is brought about bythe low exposure to a normally toxic stimuli (e.g. reactive oxygen), bystimulating and modulating stress response pathways in cells of thetargeted tissues. Endogenous response to exogenous generated freeradicals (reactive oxygen species) is modulated in increased defensecapacity against the exogenous free radicals and induces acceleration ofhealing and regenerative processes. Furthermore, activation of thecomposition can also produce an antibacterial effect. The extremesensitivity of bacteria to exposure to free radicals makes thecomposition of the present disclosure a de facto bactericidalcomposition.

As stated above, the generation of oxygen species by the composition insome embodiments is accompanied by the micro-bubbling which cancontribute to debridement or dislodging of biofilm at the site ofapplication. This can allow for the improved penetration of theactivating and/or fluorescence light to the treatment site for exampleto deactivate bacterial colonies leading to their reduction in number.

Suitable oxygen-releasing agents that may be included in the compositioninclude, but are not limited to:

Hydrogen peroxide (H₂O₂) is the starting material to prepare organicperoxides. H₂O₂ is a powerful oxygen-releasing agent, and the uniqueproperty of hydrogen peroxide is that it breaks down into water andoxygen and does not form any persistent, toxic residual compound.Hydrogen peroxide for use in this composition can be used in a gel, forexample with 6% hydrogen peroxide. A suitable range of concentrationover which hydrogen peroxide can be used in the present composition isfrom about 0.1% to about 6%.

Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxideor percarbamide) is soluble in water and contains approximately 35%hydrogen peroxide. Carbamide peroxide for use in this composition can beused as a gel, for example with 16% carbamide peroxide that represents5.6% hydrogen peroxide, or 12% carbamide peroxide. A suitable range ofconcentration over which urea peroxide can be used in the presentcomposition is from about 0.3% to about 16%. Urea peroxide breaks downto urea and hydrogen peroxide in a slow-release fashion that can beaccelerated with heat or photochemical reactions. The released urea[carbamide, (NH₂)CO₂)], is highly soluble in water and is a powerfulprotein denaturant. It increases solubility of some proteins andenhances rehydration of the skin and/or mucosa.

Benzoyl peroxide consists of two benzoyl groups (benzoic acid with the Hof the carboxylic acid removed) joined by a peroxide group. It is foundin treatments for acne, in concentrations varying from 2.5% to 10%. Thereleased peroxide groups are effective at killing bacteria. Benzoylperoxide also promotes skin turnover and clearing of pores, whichfurther contributes to decreasing bacterial counts and reduce acne.Benzoyl peroxide breaks down to benzoic acid and oxygen upon contactwith skin, neither of which is toxic. A suitable range of concentrationover which benzoyl peroxide can be used in the present composition isfrom about 2.5% to about 5%.

Specific oxygen-releasing agents that that are preferably used in thematerials or methods of this disclosure include, but are not limited tohydrogen peroxide, carbamide peroxide, or benzoyl peroxide. Peroxy acid,alkali metal peroxides, alkali metal percarbonates, peroxyacetic acid,and alkali metal perborates can also be included as the oxygen-releasingagent. Oxygen-releasing agents can be provided in powder, liquid or gelform. Alternatively, the oxygen-releasing agents may also be applied tothe tissue site separately to the composition. Alternatively, thecomposition may include an amount of oxygen-releasing agent, which isaugmented by the separate application of oxygen-releasing agents to thetreatment site.

In the compositions and methods of the present disclosure, additionalcomponents may optionally be included, or used in combination with thebiophotonic compositions as described herein. Such additional componentsinclude, but are not limited to, healing factors, growth factors,antimicrobials, wrinkle fillers (e.g. botox, hyaluronic acid orpolylactic acid), collagens, anti-virals, anti-fungals, antibiotics,drugs, and/or agents that promote collagen synthesis. These additionalcomponents may be applied to the wound, skin or mucosa in a topicalfashion, prior to, at the same time of and/or after topical applicationof the biophotonic composition of the present disclosure, and may alsobe systemically administered. Suitable healing factors, antimicrobials,collagens, and/or agents that promote collagen synthesis are discussedbelow:

(d) Healing Factors

Healing factors comprise compounds that promote or enhance the healingor regenerative process of the tissues on the application site of thecomposition. During the photoactivation of the composition of thepresent disclosure, there is an increase of the absorption of moleculesat the treatment site by the skin, wound or the mucosa. An augmentationin the blood flow at the site of treatment is observed for an extentperiod of time. An increase in the lymphatic drainage and a possiblechange in the osmotic equilibrium due to the dynamic interaction of thefree radical cascades can be enhanced or even fortified with theinclusion of healing factors. Suitable healing factors include, but arenot limited to:

Hyaluronic acid (Hyaluronan, hyaluronate): is a non-sulfatedglycosaminoglycan, distributed widely throughout connective, epithelialand neural tissues. It is one of the primary components of theextracellular matrix, and contributes significantly to cellproliferation and migration. Hyaluronan is a major component of theskin, where it is involved in tissue repair. While it is abundant inextracellular matrices, it contributes to tissues hydrodynamics,movement and proliferation of cells and participates in a wide number ofcell surface receptor interactions, notably those including primaryreceptor CD44. The hyaluronidases enzymes degrade hyaluronan. There areat least seven types of hyaluronidase-like enzymes in humans, several ofwhich are tumor suppressors. The degradation products of hyaluronicacid, the oligosaccharides and the very-low molecular weight hyaluronicacid, exhibit pro-angiogenic properties. In addition, recent studiesshow that hyaluronan fragments, but not the native high molecular massof hyaluronan, can induce inflammatory responses in macrophages anddendritic cells in tissue injury. Hyaluronic acid is well suited tobiological applications targeting the skin. Due to its highbiocompatibility, it is used to stimulate tissue regeneration. Studieshave shown hyaluronic acid appearing in the early stages of healing tophysically create room for white blood cells that mediate the immuneresponse. It is used in the synthesis of biological scaffolds for woundhealing applications and in wrinkle treatment. A suitable range ofconcentration over which hyaluronic acid can be used in the presentcomposition is from about 0.001% to about 3%.

Glucosamine: is one of the most abundant monosaccharides in humantissues and a precursor in the biological synthesis of glycosilatedproteins and lipids. It is commonly used in the treatment ofosteoarthritis. The common form of glucosamine used is its sulfate saltand including glucosamine sulfate sodium chloride. Glucosamine shows anumber of effects including an anti-inflammatory activity, stimulationof the synthesis of proteoglycans and the synthesis of proteolyticenzymes. A suitable range of concentration over which glucosamine can beused in the present composition is from about 0.01% to about 3%.

Allantoin: is a diureide of glyosilic acid. It has keratolytic effect,increases the water content of the extracellular matrix, enhances thedesquamation of the upper layers of dead (apoptotic) skin cells, andpromotes skin proliferation and wound healing.

Also, saffron can act as both a chromophore and a healing factor, and asa potentiator. Other healing agents can also be included such as growthfactors.

(e) Antimicrobials

Antimicrobials kill microbes or inhibit their growth or accumulation.Exemplary antimicrobials (or antimicrobial agent) are recited in U.S.Patent Application Publications 20040009227 and 20110081530. Suitableantimicrobials for use in the methods of the present disclosure include,but not limited to, phenolic and chlorinated phenolic and chlorinatedphenolic compounds, resorcinol and its derivatives, bisphenoliccompounds, benzoic esters (parabens), halogenated carbonilides,polymeric antimicrobial agents, thazolines, trichloromethylthioimides,natural antimicrobial agents (also referred to as “natural essentialoils”), metal salts, and broad-spectrum antibiotics.

Specific phenolic and chlorinated phenolic antimicrobial agents that canbe used in the disclosure include, but are not limited to: phenol;2-methyl phenol; 3-methyl phenol; 4-methyl phenol; 4-ethyl phenol;2,4-dimethyl phenol; 2,5-dimethyl phenol; 3,4-dimethyl phenol;2,6-dimethyl phenol; 4-n-propyl phenol; 4-n-butyl phenol; 4-n-amylphenol; 4-tert-amyl phenol; 4-n-hexyl phenol; 4-n-heptyl phenol; mono-and poly-alkyl and aromatic halophenols; p-chlorophenyl; methylp-chlorophenol; ethyl p-chlorophenol; n-propyl p-chlorophenol; n-butylp-chlorophenol; n-amyl p-chlorophenol; sec-amyl p-chlorophenol; n-hexylp-chlorophenol; cyclohexyl p-chlorophenol; n-heptyl p-chlorophenol;n-octyl; p-chlorophenol; o-chlorophenol; methyl o-chlorophenol; ethylo-chlorophenol; n-propyl o-chlorophenol; n-butyl o-chlorophenol; n-amylo-chlorophenol; tert-amyl o-chlorophenol; n-hexyl o-chlorophenol;n-heptyl o-chlorophenol; o-benzyl p-chlorophenol; o-benxyl-m-methylp-chlorophenol; o-benzyl-m,m-dimethyl p-chlorophenol; o-phenylethylp-chlorophenol; o-phenylethyl-m-methyl p-chlorophenol; 3-methylp-chlorophenol 3,5-dimethyl p-chlorophenol, 6-ethyl-3-methylp-chlorophenol, 6-n-propyl-3-methyl p-chlorophenol;6-iso-propyl-3-methyl p-chlorophenol; 2-ethyl-3,5-dimethylp-chlorophenol; 6-sec-butyl-3-methyl p-chlorophenol;2-iso-propyl-3,5-dimethyl p-chlorophenol; 6-diethylmethyl-3-methylp-chlorophenol; 6-iso-propyl-2-ethyl-3-methyl p-chlorophenol;2-sec-amyl-3,5-dimethyl p-chlorophenol; 2-diethylmethyl-3,5-dimethylp-chlorophenol; 6-sec-octyl-3-methyl p-chlorophenol; p-chloro-m-cresolp-bromophenol; methyl p-bromophenol; ethyl p-bromophenol; n-propylp-bromophenol; n-butyl p-bromophenol; n-amyl p-bromophenol; sec-amylp-bromophenol; n-hexyl p-bromophenol; cyclohexyl p-bromophenol;o-bromophenol; tert-amyl o-bromophenol; n-hexyl o-bromophenol;n-propyl-m,m-dimethyl o-bromophenol; 2-phenyl phenol; 4-chloro-2-methylphenol; 4-chloro-3-methyl phenol; 4-chloro-3,5-dimethyl phenol;2,4-dichloro-3,5-dimethylphenol; 3,4,5,6-tetabromo-2-methylphenol-;5-methyl-2-pentylphenol; 4-isopropyl-3-methylphenol;para-chloro-metaxylenol (PCMX); chlorothymol; phenoxyethanol;phenoxyisopropanol; and 5-chloro-2-hydroxydiphenylmethane.

Resorcinol and its derivatives can also be used as antimicrobial agents.Specific resorcinol derivatives include, but are not limited to: methylresorcinol; ethyl resorcinol; n-propyl resorcinol; n-butyl resorcinol;n-amyl resorcinol; n-hexyl resorcinol; n-heptyl resorcinol; n-octylresorcinol; n-nonyl resorcinol; phenyl resorcinol; benzyl resorcinol;phenylethyl resorcinol; phenylpropyl resorcinol; p-chlorobenzylresorcinol; 5-chloro-2,4-dihydroxydiphenyl methane;4′-chloro-2,4-dihydroxydiphenyl methane; 5-bromo-2,4-dihydroxydiphenylmethane; and 4′-bromo-2,4-dihydroxydiphenyl methane.

Specific bisphenolic antimicrobial agents that can be used in thedisclosure include, but are not limited to: 2,2′-methylenebis-(4-chlorophenol); 2,4,4′trichloro-2′-hydroxy-diphenyl ether, whichis sold by Ciba Geigy, Florham Park, N.J. under the tradenameTriclosan®; 2,2′-methylene bis-(3,4,6-trichlorophenol); 2,2′-methylenebis-(4-chloro-6-bromophenol); bis-(2-hydroxy-3,5-dichlorop-henyl)sulphide; and bis-(2-hydroxy-5-chlorobenzyl)sulphide.

Specific benzoic esters (parabens) that can be used in the disclosureinclude, but are not limited to: methylparaben; propylparaben;butylparaben; ethylparaben; isopropylparaben; isobutylparaben;benzylparaben; sodium methylparaben; and sodium propylparaben.

Specific halogenated carbanilides that can be used in the disclosureinclude, but are not limited to: 3,4,4′-trichlorocarbanilides, such as3-(4-chlorophenyl)-1-(3,4-dichlorphenyl)urea sold under the tradenameTriclocarban® by Ciba-Geigy, Florham Park, N.J.; 3-trifluoromethyl-4,4′-dichlorocarbanilide; and 3,3′,4-trichlorocarbanilide.

Specific polymeric antimicrobial agents that can be used in thedisclosure include, but are not limited to: polyhexamethylene biguanidehydrochloride; and poly(iminoimidocarbonyl iminoimidocarbonyliminohexamethylene hydrochloride), which is sold under the tradenameVantocil® IB.

Specific thazolines that can be used in the disclosure include, but arenot limited to that sold under the tradename Micro-Check®; and2-n-octyl-4-isothiazolin-3-one, which is sold under the tradenameVinyzene® IT-3000 DIDP.

Specific trichloromethylthioimides that can be used in the disclosureinclude, but are not limited to: N-(trichloromethylthio)phthalimide,which is sold under the tradename Fungitrol®; andN-trichloromethylthio-4-cyclohexene-1,2-dicarboximide, which is soldunder the tradename Vancide®.

Specific natural antimicrobial agents that can be used in the disclosureinclude, but are not limited to, oils of: anise; lemon; orange;rosemary; wintergreen; thyme; lavender; cloves; hops; tea tree;citronella; wheat; barley; lemongrass; cedar leaf; cedarwood; cinnamon;fleagrass; geranium; sandalwood; violet; cranberry; eucalyptus; vervain;peppermint; gum benzoin; basil; fennel; fir; balsam; menthol; ocmeaoriganuin; hydastis; carradensis; Berberidaceac daceae; Ratanhiae longa;and Curcuma longa. Also included in this class of natural antimicrobialagents are the key chemical components of the plant oils which have beenfound to provide antimicrobial benefit. These chemicals include, but arenot limited to: anethol; catechole; camphene; thymol; eugenol;eucalyptol; ferulic acid; farnesol; hinokitiol; tropolone; limonene;menthol; methyl salicylate; carvacol; terpineol; verbenone; berberine;ratanhiae extract; caryophellene oxide; citronellic acid; curcumin;nerolidol; and geraniol.

Specific metal salts that can be used in the disclosure include, but arenot limited to, salts of metals in groups 3a-5a, 3b-7b, and 8 of theperiodic table. Specific examples of metal salts include, but are notlimited to, salts of: aluminum; zirconium; zinc; silver; gold; copper;lanthanum; tin; mercury; bismuth; selenium; strontium; scandium;yttrium; cerium; praseodymiun; neodymium; promethum; samarium; europium;gadolinium; terbium; dysprosium; holmium; erbium; thalium; ytterbium;lutetium; and mixtures thereof. An example of the metal-ion basedantimicrobial agent is sold under the tradename HealthShield®, and ismanufactured by HealthShield Technology, Wakefield, Mass. [give otherexamples here e.g. smith and nephew]

Specific broad-spectrum antimicrobial agents that can be used in thedisclosure include, but are not limited to, those that are recited inother categories of antimicrobial agents herein.

Additional antimicrobial agents that can be used in the methods of thedisclosure include, but are not limited to: pyrithiones, and inparticular pyrithione-including zinc complexes such as that sold underthe tradename Octopirox®; dimethyidimethylol hydantoin, which is soldunder the tradename Glydant®;methylchloroisothiazolinone/methylisothiazolinone, which is sold underthe tradename Kathon CG®; sodium sulfite; sodium bisulfite;imidazolidinyl urea, which is sold under the tradename Germall 115®;diazolidinyl urea, which is sold under the tradename Germall 110; benzylalcohol v2-bromo-2-nitropropane-1,3-diol, which is sold under thetradename Bronopol®; formalin or formaldehyde; iodopropenylbutylcarbamate, which is sold under the tradename Polyphase P100®;chloroacetamide; methanamine; methyldibromonitrile glutaronitrile(1,2-dibromo-2,4-dicyanobutane), which is sold under the tradenameTektamer®; glutaraldehyde; 5-bromo-5-nitro-1,3-dioxane, which is soldunder the tradename Bronidox®; phenethyl alcohol; o-phenylphenol/sodiumo-phenylphenol sodium hydroxymethylglycinate, which is sold under thetradename Suttocide A®; polymethoxy bicyclic oxazolidine; which is soldunder the tradename Nuosept C®; dimethoxane; thimersal; dichlorobenzylalcohol; captan; chlorphenenesin; dichlorophene; chlorbutanol; glyceryllaurate; halogenated diphenyl ethers;2,4,4′-trichloro-2′-hydroxy-diphenyl ether, which is sold under thetradename Triclosan® and is available from Ciba-Geigy, Florham Park,N.J.; and 2,2′-dihydroxy-5,5′-dibromo-diphenyl ether.

Additional antimicrobial agents that can be used in the methods of thedisclosure include those disclosed by U.S. Pat. Nos. 3,141,321;4,402,959; 4,430,381; 4,533,435; 4,625,026; 4,736,467; 4,855,139;5,069,907; 5,091,102; 5,639,464; 5,853,883; 5,854,147; 5,894,042; and5,919,554, and U.S. Pat. Appl. Publ. Nos. 20040009227 and 20110081530.

(f) Collagens and Agents that Promote Collagen Synthesis

Collagen is a fibrous protein produced in dermal fibroblast cells andforming 70% of the dermis. Collagen is responsible for the smoothing andfirming of the skin. Therefore, when the synthesis of collagen isreduced, skin aging will occur, and so the firming and smoothing of theskin will be rapidly reduced. As a result, the skin will be flaccid andwrinkled. On the other hand, when metabolism of collagen is activated bythe stimulation of collagen synthesis in the skin, the components ofdermal matrices will be increased, leading to effects, such as wrinkleimprovement, firmness improvement and skin strengthening. Thus,collagens and agents that promote collagen synthesis may also be usefulin the present disclosure. Agents that promote collagen synthesis (i.e.,pro-collagen synthesis agents) include amino acids, peptides, proteins,lipids, small chemical molecules, natural products and extracts fromnatural products.

For instance, it was discovered that intake of vitamin C, iron, andcollagen can effectively increase the amount of collagen in skin orbone. See, e.g., U.S. Patent Application Publication 20090069217.Examples of the vitamin C include an ascorbic acid derivative such asL-ascorbic acid or sodium L-ascorbate, an ascorbic acid preparationobtained by coating ascorbic acid with an emulsifier or the like, and amixture containing two or more of those vitamin Cs at an arbitrary rate.In addition, natural products containing vitamin C such as acerola andlemon may also be used. Examples of the iron preparation include: aninorganic iron such as ferrous sulfate, sodium ferrous citrate, orferric pyrophosphate; an organic iron such as heme iron, ferritin iron,or lactoferrin iron; and a mixture containing two or more of those ironsat an arbitrary rate. In addition, natural products containing iron suchas spinach or liver may also be used. Moreover, examples of the collageninclude: an extract obtained by treating bone, skin, or the like of amammal such as bovine or swine with an acid or alkaline; a peptideobtained by hydrolyzing the extract with a protease such as pepsine,trypsin, or chymotrypsin; and a mixture containing two or more of thosecollagens at an arbitrary rate. Collagens extracted from plant sourcesmay also be used.

Additional pro-collagen synthesis agents are described, for example, inU.S. Pat. Nos. 7,598,291, 7,722,904, 6,203,805, 5,529,769, etc, and U.S.Patent Application Publications 20060247313, 20080108681, 20110130459,20090325885, 20110086060, etc.

The composition can also include other ingredients such as humectants(e.g. glycerine and propylene glycol), preservatives such as parabens,and pH adjusters such as sodium hydroxide.

(4) Methods of Use

The biophotonic compositions of the present disclosure have numeroususes. Without being bound by theory, the biophotonic compositions of thepresent disclosure may promote wound healing or tissue repair. Thebiophotonic compositions of the present disclosure may also be used totreat a skin disorder. The biophotonic compositions of the presentdisclosure may also be used to treat acne. The biophotonic compositionsof the present disclosure may also be used for skin rejuvenation. Thebiophotonic compositions of the present disclosure may also be used fortreating acute inflammation. Therefore, it is an objective of thepresent disclosure to provide a method for providing biophotonic therapyto a wound, where the method promotes wound healing. It is also anobjective of the present disclosure to provide a method for providingbiophotonic therapy to a skin tissue afflicted with acne, wherein themethod is used to treat acne. It is also an objective of the presentdisclosure to provide a method for providing biophotonic therapy to askin tissue afflicted with a skin disorder, wherein the method is usedto treat the skin disorder. It is also an objective of the presentdisclosure to provide a method for providing biophotonic therapy to skintissue, wherein the method is used for promoting skin rejuvenation.

In certain embodiments, the present disclosure provides a method forproviding a biophotonic therapy to a wound, the method comprising:applying (e.g., by topical application) a biophotonic composition of thepresent disclosure to a site of a wound, and illuminating thebiophotonic composition with light having a wavelength that overlapswith an absorption spectrum of the chromophore(s) of the biophotoniccomposition.

In one aspect, the present disclosure provides a method for providingbiophotonic therapy to a wound, comprising: topically applying abiophotonic composition comprising a first chromophore; and illuminatingsaid biophotonic composition with light having a wavelength thatoverlaps with an absorption spectrum of the first chromophore; whereinthe biophotonic composition is substantially resistant to leaching suchthat it limits leaching of the chromophore into the tissue duringtreatment. In some embodiments, less than 30%, 25%, 20%, 15%, 10%, 5%,1%, 0.8%, 0.5% or 0.1% or essentially 0% of the total chromophore amountleaches out of the biophotonic composition into the wound or tissueduring treatment.

In another aspect, the present disclosure provides a method for treatinga wound or providing biophotonic therapy to a wound, comprising:topically applying a biophotonic composition comprising a firstchromophore and a gelling agent to a site of a wound; and illuminatingsaid biophotonic composition with light having a wavelength thatoverlaps with an absorption spectrum of the first chromophore; whereinthe gelling agent blocks substantial leaching of the chromophores intothe site of a wound during treatment. In some embodiments, less than30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or 0.1% or essentially 0% ofthe total chromophore amount leaches out of the biophotonic compositioninto the wound or tissue during treatment.

In yet another aspect, the present disclosure provides a method forpromoting skin rejuvenation. In certain embodiments, the presentdisclosure provides a method for providing skin rejuvenation, the methodcomprising: applying (e.g., by topical application) a biophotoniccomposition of the present disclosure to the skin, and illuminating thebiophotonic composition with light having a wavelength that overlapswith an absorption spectrum of the chromophore(s) of the biophotoniccomposition.

In other embodiments, the present disclosure provides a method forpromoting skin rejuvenation comprising: topically applying a biophotoniccomposition comprising a first chromophore to skin; and illuminatingsaid biophotonic composition with light having a wavelength thatoverlaps with an absorption spectrum of the first chromophore; whereinthe biophotonic composition is substantially resistant to leaching suchthat it limits leaching of the chromophore into the skin duringtreatment. In some embodiments, less than 30%, 25%, 20%, 15%, 10%, 5%,1%, 0.8%, 0.5% or 0.1% or essentially 0% of the total chromophore amountleaches out of the biophotonic composition into the wound or tissueduring treatment.

In another aspect, the present disclosure provides a method forpromoting skin rejuvenation, comprising: topically applying abiophotonic composition comprising a first chromophore and a gellingagent to skin; and illuminating said biophotonic composition with lighthaving a wavelength that overlaps with an absorption spectrum of thefirst chromophore; wherein the biophotonic composition is substantiallyresistant to leaching such that it blocks substantial leaching of thechromophores into the skin during treatment. In some embodiments, lessthan 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or 0.1% or essentially0% of the total chromophore amount leaches out of the biophotoniccomposition into the skin during treatment.

In yet another aspect, the present disclosure to provide a method forproviding biophotonic therapy to a target skin tissue afflicted with askin disorder. In certain embodiments, the present disclosure provides amethod for providing a biophotonic therapy to a target skin tissue, themethod comprising: applying (e.g., by topical application) a biophotoniccomposition of the present disclosure to a target skin tissue, andilluminating the biophotonic composition with light having a wavelengththat overlaps with an absorption spectrum of the chromophore(s) of thebiophotonic composition.

In other embodiments, the present disclosure provides a method fortreating a skin disorder, comprising: topically applying a biophotoniccomposition to a target skin tissue afflicted with the skin disorder,wherein the biophotonic composition comprises a first chromophore; andilluminating said biophotonic composition with light having a wavelengththat overlaps with an absorption spectrum of the first chromophore;wherein the biophotonic composition is substantially resistant toleaching such that it limits leaching of the chromophore into the skinduring treatment. In some embodiments, less than 30%, 25%, 20%, 15%,10%, 5%, 1%, 0.8%, 0.5% or 0.1% or essentially 0% of the totalchromophore amount leaches out of the biophotonic composition into theskin during treatment.

In another aspect, the present disclosure provides a method for treatinga skin disorder, comprising: topically applying a biophotoniccomposition comprising a first chromophore and a gelling agent to skinafflicted with the skin disorder; and illuminating said biophotoniccomposition with light having a wavelength that overlaps with anabsorption spectrum of the first chromophore; wherein the biophotoniccomposition is substantially resistant to leaching such that it blockssubstantial leaching of the chromophores into the skin during treatment.In some embodiments, less than 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%,0.5% or 0.1% or essentially 0% of the total chromophore amount leachesout of the biophotonic composition into the skin during treatment.

In yet another aspect, the present disclosure to provide a method forproviding biophotonic therapy to a target skin tissue afflicted withacne. In certain embodiments, the present disclosure provides a methodfor providing a biophotonic therapy to a target skin tissue afflictedwith acne, the method comprising: applying (e.g., by topicalapplication) a biophotonic composition of the present disclosure to atarget skin tissue, and illuminating the biophotonic composition withlight having a wavelength that overlaps with an absorption spectrum ofthe chromophore(s) of the biophotonic composition.

In other embodiments, the present disclosure provides a method fortreating acne, comprising: topically applying a biophotonic compositionto a target skin tissue afflicted with acne, wherein the biophotoniccomposition comprises a first chromophore; illuminating said biophotoniccomposition with light having a wavelength that overlaps with anabsorption spectrum of the first chromophore; wherein the biophotoniccomposition is substantially resistant to leaching such that it limitsleaching of the chromophore into tissue during treatment. In someembodiments, less than 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or0.1% or essentially 0% of the total chromophore amount leaches out ofthe biophotonic composition into the tissue during treatment.

In another aspect, the present disclosure provides a method for treatingacne, comprising: topically applying a biophotonic compositioncomprising a first chromophore to skin afflicted with acne; andilluminating said biophotonic composition with light having a wavelengththat overlaps with an absorption spectrum of the first chromophore;wherein the biophotonic composition is substantially resistant toleaching such that it blocks substantial leaching of the chromophoresinto the skin during treatment. In some embodiments, less than 30%, 25%,20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or 0.1% or essentially 0% of the totalchromophore amount leaches out of the biophotonic composition into thewound or tissue during treatment.

In other embodiments, the present disclosure provides a method fortreating acute inflammation, comprising: topically applying abiophotonic composition to a target skin tissue with acute inflammation,wherein the biophotonic composition comprises a first chromophore;illuminating said biophotonic composition with light having a wavelengththat overlaps with an absorption spectrum of the first chromophore;wherein the biophotonic composition is substantially resistant toleaching such that it limits leaching of the chromophore into tissueduring treatment. In some embodiments, less than 30%, 25%, 20%, 15%,10%, 5%, 1%, 0.8%, 0.5% or 0.1% or essentially 0% of the totalchromophore amount leaches out of the biophotonic composition into thetissue during treatment.

In another aspect, the present disclosure provides a method for treatingacute inflammation, comprising: topically applying a biophotoniccomposition comprising a first chromophore to skin afflicted with acuteinflammation; and illuminating said biophotonic composition with lighthaving a wavelength that overlaps with an absorption spectrum of thefirst chromophore; wherein the biophotonic composition is substantiallyresistant to leaching such that it blocks substantial leaching of thechromophores into the skin during treatment. In some embodiments, lessthan 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5% or 0.1% or essentially0% of the total chromophore amount leaches out of the biophotoniccomposition into the wound or tissue during treatment.

In another aspect, the present disclosure provides a method for treatingfungal infections, comprising: topically applying a biophotoniccomposition comprising a first chromophore to a target site afflictedwith acute inflammation; and illuminating said biophotonic compositionwith light having a wavelength that overlaps with an absorption spectrumof the first chromophore; wherein the biophotonic composition issubstantially resistant to leaching such that it blocks substantialleaching of the chromophores into the target site during treatment. Insome embodiments, less than 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.8%, 0.5%or 0.1% or essentially 0% of the total chromophore amount leaches out ofthe biophotonic composition into the wound or tissue during treatment.In some embodiments, the target site may be skin or nails.

The biophotonic compositions suitable for use in the methods of thepresent disclosure may be selected from any of the embodiments of thebiophotonic compositions described above. For instance, the biophotoniccompositions useful in the method of the present disclosure may comprisea first chromophore that undergoes at least partial photobleaching uponapplication of light. The first chromophore may absorb at a wavelengthof about 200-800 nm, 200-700 nm, 200-600 nm or 200-500 nm. In oneembodiment, the first chromophore absorbs at a wavelength of about200-600 nm. In some embodiments, the first chromophore absorbs light ata wavelength of about 200-300 nm, 250-350 nm, 300-400 nm, 350-450 nm,400-500 nm, 450-650 nm, 600-700 nm, 650-750 nm or 700-800 nm. In otherexamples, suitable biophotonic compositions for the methods of thepresent disclosure may further comprise at least one additionalchromophore (e.g., a second chromophore). The absorption spectrum of thesecond chromophore overlaps at least about 80%, 50%, 40%, 30%, or 20%with the emission spectrum of the first chromophore. In someembodiments, the first chromophore has an emission spectrum thatoverlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%,35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of thesecond chromophore.

Illumination of the biophotonic composition with light may cause atransfer of energy from the first chromophore to the second chromophore.Subsequently, the second chromophore may emit energy as fluorescenceand/or generate reactive oxygen species. In certain embodiments of themethods the present disclosure, energy transfer caused by theapplication of light is not accompanied by concomitant generation ofheat, or does not result in tissue damage.

The biophotonic compositions useful for the present methods comprise agelling agent. The gelling agent may include, but is not limited to,lipids such as glycerin, glycols such as propylene glycol, hyaluronicacid, glucosamine sulfate, cellulose derivatives (hydroxypropylmethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,methylcellulose and the like), noncellulose polysaccharides(galactomannans, guar gum, carob gum, gum arabic, sterculia gum, agar,alginates and the like) and acrylic acid polymers.

When the method involves a biophotonic composition having at least twochromophores, the first chromophore is present in an amount of about0.01-40% per weight of the composition, and the second chromophore ispresent in an amount of about 0.001-40% per weight of the composition.In certain embodiments, the first chromophore is present in an amount ofabout 0.01-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%,12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%,30-35%, 32.5-37.5%, or 35-40% per weight of the composition. In certainembodiments, the second chromophore is present in an amount of about0.001-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%,15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%,32.5-37.5%, or 35-40% per weight of the composition. In certainembodiments, the total weight per weight of chromophore or combinationof chromophores may be in the amount of about 0.01-1%, 0.5-2%, 1-5%,2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%,20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40.05%per weight of the composition.

In the methods of the present disclosure, any source of actinic lightcan be used. Any type of halogen, LED or plasma arc lamp or laser may besuitable. The primary characteristic of suitable sources of actiniclight will be that they emit light in a wavelength (or wavelengths)appropriate for activating the one or more photoactivators present inthe composition. In one embodiment, an argon laser is used. In anotherembodiment, a potassium-titanyl phosphate (KTP) laser (e.g. aGreenLight™ laser) is used. In another embodiment, sunlight may be used.In yet another embodiment, a LED photocuring device is the source of theactinic light. In yet another embodiment, the source of the actiniclight is a source of light having a wavelength between about 200 to 800nm. In another embodiment, the source of the actinic light is a sourceof visible light having a wavelength between about 400 and 600 nm.Furthermore, the source of actinic light should have a suitable powerdensity. Suitable power density for non-collimated light sources (LED,halogen or plasma lamps) are in the range from about 1 mW/cm² to about200 mW/cm². Suitable power density for laser light sources are in therange from about 0.5 mW/cm² to about 0.8 mW/cm².

In some embodiments of the methods of the present disclosure, the lighthas an energy at the subject's skin, wound or mucosa surface of betweenabout 1 mW/cm² and about 500 mW/cm², 1-300 mW/cm², or 1-200 mW/cm²,wherein the energy applied depends at least on the condition beingtreated, the wavelength of the light, the distance of the subject's skinfrom the light source, and the thickness of the biophotonic composition.In certain embodiments, the light at the subject's skin is between about1-40 mW/cm², or 20-60 mW/cm², or 40-80 mW/cm², or 60-100 mW/cm², or80-120 mW/cm², or 100-140 mW/cm², or 120-160 mW/cm², or 140-180 mW/cm²,or 160-200 mW/cm², or 110-240 mW/cm², or 110-150 mW/cm², or 190-240mW/cm².

In some embodiments, a mobile device can be used to activate embodimentsof the biophotonic composition of the present disclosure, wherein themobile device can emit light having an emission spectra which overlapsan absorption spectra of the chromophore in the biophotonic composition.The mobile device can have a display screen through which the light isemitted and/or the mobile device can emit light from a flashlight whichcan photoactivate the biophotonic composition.

In some embodiments, a display screen on a television or a computermonitor can be used to activate the biophotonic composition, wherein thedisplay screen can emit light having an emission spectra which overlapsan absorption spectra of a photoactive agent in the photoactivatablecomposition.

In certain embodiments, the first and/or the second chromophore (whenpresent) can be photoactivated by ambient light which may originate fromthe sun or other light sources. Ambient light can be considered to be ageneral illumination that comes from all directions in a room that hasno visible source. In certain embodiments, the first and/or the secondchromophore (when present) can be photoactivated by light in the visiblerange of the electromagnetic spectrum. Exposure times to ambient lightmay be longer than that to direct light.

In certain embodiments, different sources of light can be used toactivate the biophotonic compositions, such as a combination of ambientlight and direct LED light.

The duration of the exposure to actinic light required will be dependenton the surface of the treated area, the type of lesion, trauma or injurythat is being treated, the power density, wavelength and bandwidth ofthe light source, the thickness of the biophotonic composition, and thetreatment distance from the light source. The illumination of thetreated area by fluorescence may take place within seconds or evenfragment of seconds, but a prolonged exposure period is beneficial toexploit the synergistic effects of the absorbed, reflected and reemittedlight on the composition of the present disclosure and its interactionwith the tissue being treated. In one embodiment, the time of exposureto actinic light of the tissue, skin or wound on which the biophotoniccomposition has been applied is a period between 1 minute and 5 minutes.In another embodiment, the time of exposure to actinic light of thetissue, skin or wound on which the biophotonic composition has beenapplied is a period between 1 minute and 5 minutes. In some otherembodiments, the biophotonic composition is illuminated for a periodbetween 1 minute and 3 minutes. In certain embodiments, light is appliedfor a period of 1-30 seconds, 15-45 seconds, 30-60 seconds, 0.75-1.5minutes, 1-2 minutes, 1.5-2.5 minutes, 2-3 minutes, 2.5-3.5 minutes, 3-4minutes, 3.5-4.5 minutes, 4-5 minutes, 5-10 minutes, 10-15 minutes,15-20 minutes, 20-25 minutes, or 20-30 minutes. In yet anotherembodiment, the source of actinic light is in continuous motion over thetreated area for the appropriate time of exposure. In yet anotherembodiment, multiple applications of the biophotonic composition andactinic light are performed. In some embodiments, the tissue, skin orwound is exposed to actinic light at least two, three, four, five or sixtimes. In some embodiments, a fresh application of the biophotoniccomposition is applied before exposure to actinic light.

In the methods of the present disclosure, the biophotonic compositionmay be optionally removed from the site of treatment followingapplication of light. In certain embodiments, the biophotoniccomposition is left on the treatment site for more than 30 minutes, morethan one hour, more than 2 hours, more than 3 hours. It can beilluminated with ambient light. To prevent drying, the composition canbe covered with a transparent or translucent cover such as a polymerfilm, or an opaque cover which can be removed before illumination.

(5) Wounds and Wound Healing

The biophotonic compositions and methods of the present disclosure maybe used to treat wounds and promote wound healing. Wounds that may betreated by the biophotonic compositions and methods of the presentdisclosure include, for example, injuries to the skin and subcutaneoustissue initiated in different ways (e.g., pressure ulcers from extendedbed rest, wounds induced by trauma, wounds induced by conditions such asperiodontitis) and with varying characteristics. In certain embodiments,the present disclosure provides biophotonic compositions and methods fortreating and/or promoting the healing of, for example, burns, incisions,excisions, lacerations, abrasions, puncture or penetrating wounds,surgical wounds, contusions, hematomas, crushing injuries, sores andulcers.

Biophotonic compositions and methods of the present disclosure may beused to treat and/or promote the healing of chronic cutaneous ulcers orwounds, which are wounds that have failed to proceed through an orderlyand timely series of events to produce a durable structural, functional,and cosmetic closure. The vast majority of chronic wounds can beclassified into three categories based on their etiology: pressureulcers, neuropathic (diabetic foot) ulcers and vascular (venous orarterial) ulcers.

In certain other embodiments, the present disclosure providesbiophotonic compositions and methods for treating and/or promotinghealing, Grade I-IV ulcers. In certain embodiments, the applicationprovides compositions suitable for use with Grade II ulcers inparticular. Ulcers may be classified into one of four grades dependingon the depth of the wound: i) Grade I: wounds limited to the epithelium;ii) Grade II: wounds extending into the dermis; iii) Grade III: woundsextending into the subcutaneous tissue; and iv) Grade IV (orfull-thickness wounds): wounds wherein bones are exposed (e.g., a bonypressure point such as the greater trochanter or the sacrum).

For example, the present disclosure provides biophotonic compositionsand methods for treating and/or promoting healing of a diabetic ulcer.Diabetic patients are prone to foot and other ulcerations due to bothneurologic and vascular complications. Peripheral neuropathy can causealtered or complete loss of sensation in the foot and/or leg. Diabeticpatients with advanced neuropathy lose all ability for sharp-dulldiscrimination. Any cuts or trauma to the foot may go completelyunnoticed for days or weeks in a patient with neuropathy. A patient withadvanced neuropathy loses the ability to sense a sustained pressureinsult, as a result, tissue ischemia and necrosis may occur leading tofor example, plantar ulcerations. Microvascular disease is one of thesignificant complications for diabetics which may also lead toulcerations. In certain embodiments, compositions and methods oftreating a chronic wound are provided here in, where the chronic woundis characterized by diabetic foot ulcers and/or ulcerations due toneurologic and/or vascular complications of diabetes.

In other examples, the present disclosure provides biophotoniccompositions and methods for treating and/or promoting healing of apressure ulcer. Pressure ulcer includes bed sores, decubitus ulcers andischial tuberosity ulcers and can cause considerable pain and discomfortto a patient. A pressure ulcer can occur as a result of a prolongedpressure applied to the skin. Thus, pressure can be exerted on the skinof a patient due to the weight or mass of an individual. A pressureulcer can develop when blood supply to an area of the skin is obstructedor cut off for more than two or three hours. The affected skin area canturns red, becomes painful and can become necrotic. If untreated, theskin breaks open and can become infected. An ulcer sore is therefore askin ulcer that occurs in an area of the skin that is under pressurefrom e.g. lying in bed, sitting in a wheelchair, and/or wearing a castfor a prolonged period of time. Pressure ulcer can occur when a personis bedridden, unconscious, unable to sense pain, or immobile. Pressureulcer often occur in boney prominences of the body such as the buttocksarea (on the sacrum or iliac crest), or on the heels of foot.

In other examples, the present disclosure provides biophotoniccompositions and methods for treating and/or promoting healing of acutewounds.

Additional types of wound that can be treated by the biophotoniccompositions and methods of the present disclosure include thosedisclosed by U.S. Pat. Appl. Publ. No. 20090220450, which isincorporated herein by reference.

Wound healing in adult tissues is a complicated reparative process. Forexample, the healing process for skin involves the recruitment of avariety of specialized cells to the site of the wound, extracellularmatrix and basement membrane deposition, angiogenesis, selectiveprotease activity and re-epithelialization.

There are three distinct phases in the wound healing process. First, inthe inflammatory phase, which typically occurs from the moment a woundoccurs until the first two to five days, platelets aggregate to depositgranules, promoting the deposit of fibrin and stimulating the release ofgrowth factors. Leukocytes migrate to the wound site and begin to digestand transport debris away from the wound. During this inflammatoryphase, monocytes are also converted to macrophages, which release growthfactors for stimulating angiogenesis and the production of fibroblasts.

Second, in the proliferative phase, which typically occurs from two daysto three weeks, granulation tissue forms, and epithelialization andcontraction begin. Fibroblasts, which are key cell types in this phase,proliferate and synthesize collagen to fill the wound and provide astrong matrix on which epithelial cells grow. As fibroblasts producecollagen, vascularization extends from nearby vessels, resulting ingranulation tissue. Granulation tissue typically grows from the base ofthe wound. Epithelialization involves the migration of epithelial cellsfrom the wound surfaces to seal the wound. Epithelial cells are drivenby the need to contact cells of like type and are guided by a network offibrin strands that function as a grid over which these cells migrate.Contractile cells called myofibroblasts appear in wounds, and aid inwound closure. These cells exhibit collagen synthesis and contractility,and are common in granulating wounds.

Third, in the remodeling phase, the final phase of wound healing whichcan take place from three weeks up to several years, collagen in thescar undergoes repeated degradation and re-synthesis. During this phase,the tensile strength of the newly formed skin increases.

However, as the rate of wound healing increases, there is often anassociated increase in scar formation. Scarring is a consequence of thehealing process in most adult animal and human tissues. Scar tissue isnot identical to the tissue which it replaces, as it is usually ofinferior functional quality. The types of scars include, but are notlimited to, atrophic, hypertrophic and keloidal scars, as well as scarcontractures. Atrophic scars are flat and depressed below thesurrounding skin as a valley or hole. Hypertrophic scars are elevatedscars that remain within the boundaries of the original lesion, andoften contain excessive collagen arranged in an abnormal pattern.Keloidal scars are elevated scars that spread beyond the margins of theoriginal wound and invade the surrounding normal skin in a way that issite specific, and often contain whorls of collagen arranged in anabnormal fashion.

In contrast, normal skin consists of collagen fibers arranged in abasket-weave pattern, which contributes to both the strength andelasticity of the dermis. Thus, to achieve a smoother wound healingprocess, an approach is needed that not only stimulates collagenproduction, but also does so in a way that reduces scar formation.

The biophotonic compositions and methods of the present disclosurepromote the wound healing by promoting the formation of substantiallyuniform epithelialization; promoting collagen synthesis; promotingcontrolled contraction; and/or by reducing the formation of scar tissue.In certain embodiments, the biophotonic compositions and methods of thepresent disclosure may promote wound healing by promoting the formationof substantially uniform epithelialization. In some embodiments, thebiophotonic compositions and methods of the present disclosure promotecollagen synthesis. In some other embodiments, the biophotoniccompositions and methods of the present disclosure promote controlledcontraction. In certain embodiments, the biophotonic compositions andmethods of the present disclosure promote wound healing, for example, byreducing the formation of scar tissue or by speeding up the woundclosure process. In certain embodiments, the biophotonic compositionsand methods of the present disclosure promote wound healing, forexample, by reducing inflammation. In certain embodiments, thebiophotonic composition can be used following wound closure to optimizescar revision. In this case, the biophotonic composition may be appliedat regular intervals such as once a week, or at an interval deemedappropriate by the physician.

The biophotonic composition may be soaked into a woven or non-wovenmaterial or a sponge and applied as a wound dressing. A light source,such as LEDs or waveguides, may be provided within or adjacent the wounddressing or the composition to illuminate the composition. Thewaveguides can be optical fibres which can transmit light, not only fromtheir ends, but also from their body. For example, made of polycarbonateor polymethylmethacrylate.

Adjunct therapies which may be topical or systemic such as antibiotictreatment may also be used. Negative pressure assisted wound closure canalso be used to assist wound closure and/or to remove the composition.

(6) Acne and Acne Scars

The biophotonic compositions and methods of the present disclosure maybe used to treat acne. As used herein, “acne” means a disorder of theskin caused by inflammation of skin glands or hair follicles. Thebiophotonic compositions and methods of the disclosure can be used totreat acne at early pre-emergent stages or later stages where lesionsfrom acne are visible. Mild, moderate and severe acne can be treatedwith embodiments of the biophotonic compositions and methods. Earlypre-emergent stages of acne usually begin with an excessive secretion ofsebum or dermal oil from the sebaceous glands located in thepilosebaceous apparatus. Sebum reaches the skin surface through the ductof the hair follicle. The presence of excessive amounts of sebum in theduct and on the skin tends to obstruct or stagnate the normal flow ofsebum from the follicular duct, thus producing a thickening andsolidification of the sebum to create a solid plug known as a comedone.In the normal sequence of developing acne, hyperkeratinazation of thefollicular opening is stimulated, thus completing blocking of the duct.The usual results are papules, pustules, or cysts, often contaminatedwith bacteria, which cause secondary infections. Acne is characterizedparticularly by the presence of comedones, inflammatory papules, orcysts. The appearance of acne may range from slight skin irritation topitting and even the development of disfiguring scars. Accordingly, thebiophotonic compositions and methods of the present disclosure can beused to treat one or more of skin irritation, pitting, development ofscars, comedones, inflammatory papules, cysts, hyperkeratinazation, andthickening and hardening of sebum associated with acne.

The composition may be soaked into or applied to a woven or non-wovenmaterial or a sponge and applied as a mask to body parts such as theface, body, arms, legs etc. A light source, such as LEDs or waveguides,may be provided within or adjacent the mask or the composition toilluminate the composition. The waveguides can be optical fibres whichcan transmit light, not only from their ends, but also from their body.For example, made of polycarbonate or polymethylmethacrylate.

The biophotonic compositions and methods of the present disclosure maybe used to treat various types of acne. Some types of acne include, forexample, acne vulgaris, cystic acne, acne atrophica, bromide acne,chlorine acne, acne conglobata, acne cosmetica, acne detergicans,epidemic acne, acne estivalis, acne fulminans, halogen acne, acneindurata, iodide acne, acne keloid, acne mechanica, acne papulosa,pomade acne, premenstral acne, acne pustulosa, acne scorbutica, acnescrofulosorum, acne urticata, acne varioliformis, acne venenata,propionic acne, acne excoriee, gram negative acne, steroid acne, andnodulocystic acne.

(7) Skin Aging and Rejuvenation

The dermis is the second layer of skin, containing the structuralelements of the skin, the connective tissue. There are various types ofconnective tissue with different functions. Elastin fibers give the skinits elasticity, and collagen gives the skin its strength.

The junction between the dermis and the epidermis is an importantstructure. The dermal-epidermal junction interlocks forming finger-likeepidermal ridges. The cells of the epidermis receive their nutrientsfrom the blood vessels in the dermis. The epidermal ridges increase thesurface area of the epidermis that is exposed to these blood vessels andthe needed nutrients.

The aging of skin comes with significant physiological changes to theskin. The generation of new skin cells slows down, and the epidermalridges of the dermal-epidermal junction flatten out. While the number ofelastin fibers increases, their structure and coherence decrease. Alsothe amount of collagen and the thickness of the dermis decrease with theageing of the skin.

Collagen is a major component of the skin's extracellular matrix,providing a structural framework. During the aging process, the decreaseof collagen synthesis and insolubilization of collagen fibers contributeto a thinning of the dermis and loss of the skin's biomechanicalproperties.

The physiological changes to the skin result in noticeable agingsymptoms often referred to as chronological-, intrinsic- andphoto-ageing. The skin becomes drier, roughness and scaling increase,the appearance becomes duller, and most obviously fine lines andwrinkles appear. Other symptoms or signs of skin aging include, but arenot limited to, thinning and transparent skin, loss of underlying fat(leading to hollowed cheeks and eye sockets as well as noticeable lossof firmness on the hands and neck), bone loss (such that bones shrinkaway from the skin due to bone loss, which causes sagging skin), dryskin (which might itch), inability to sweat sufficiently to cool theskin, unwanted facial hair, freckles, age spots, spider veins, rough andleathery skin, fine wrinkles that disappear when stretched, loose skin,a blotchy complexion.

The dermal-epidermal junction is a basement membrane that separates thekeratinocytes in the epidermis from the extracellular matrix, which liesbelow in the dermis. This membrane consists of two layers: the basallamina in contact with the keratinocytes, and the underlying reticularlamina in contact with the extracellular matrix. The basal lamina isrich in collagen type IV and laminin, molecules that play a role inproviding a structural network and bioadhesive properties for cellattachment.

Laminin is a glycoprotein that only exists in basement membranes. It iscomposed of three polypeptide chains (alpha, beta and gamma) arranged inthe shape of an asymmetric cross and held together by disulfide bonds.The three chains exist as different subtypes which result in twelvedifferent isoforms for laminin, including Laminin-1 and Laminin-5.

The dermis is anchored to hemidesmosomes, specific junction pointslocated on the keratinocytes, which consist of α-integrins and otherproteins, at the basal membrane keratinocytes by type VII collagenfibrils. Laminins, and particularly Laminin-5, constitute the realanchor point between hemidesmosomal transmembrane proteins in basalkeratinocytes and type VII collagen.

Laminin-5 synthesis and type VII collagen expression have been proven todecrease in aged skin. This causes a loss of contact between dermis andepidermis, and results in the skin losing elasticity and becoming saggy.

Recently another type of wrinkles generally referred to as expressionwrinkles, got general recognition. These wrinkles require loss ofresilience, particularly in the dermis, because of which the skin is nolonger able to resume its original state when facial muscles whichproduce facial expressions exert stress on the skin, resulting inexpression wrinkles.

The compositions and methods of the present disclosure promote skinrejuvenation. In certain embodiments, the compositions and methods ofthe present disclosure promote collagen synthesis. In certain otherembodiments, the compositions and methods of the present disclosure mayreduce, diminish, retard or even reverse one or more signs of skin agingincluding, but not limited to, appearance of fine lines or wrinkles,thin and transparent skin, loss of underlying fat (leading to hollowedcheeks and eye sockets as well as noticeable loss of firmness on thehands and neck), bone loss (such that bones shrink away from the skindue to bone loss, which causes sagging skin), dry skin (which mightitch), inability to sweat sufficiently to cool the skin, unwanted facialhair, freckles, age spots, spider veins, rough and leathery skin, finewrinkles that disappear when stretched, loose skin, or a blotchycomplexion. In certain embodiments, the compositions and methods of thepresent disclosure may induce a reduction in pore size, enhancesculpturing of skin subsections, and/or enhance skin translucence.

(8) Skin Disorders

The biophotonic compositions and methods of the present disclosure maybe used to treat skin disorders that include, but are not limited to,erythema, telangiectasia, actinic telangiectasia, psoriasis, skincancer, pemphigus, sunburn, dermatitis, eczema, rashes, impetigo, lichensimplex chronicus, rhinophyma, perioral dermatitis, pseudofolliculitisbarbae, drug eruptions, erythema multiforme, erythema nodosum, granulomaannulare, actinic keratosis, purpura, alopecia areata, aphthousstomatitis, drug eruptions, dry skin, chapping, xerosis, ichthyosisvulgaris, fungal infections, parasitic infection, herpes simplex,intertrigo, keloids, keratoses, milia, moluscum contagiosum, pityriasisrosea, pruritus, urticaria, and vascular tumors and malformations.Dermatitis includes contact dermatitis, atopic dermatitis, seborrheicdermatitis, nummular dermatitis, generalized exfoliative dermatitis, andstatis dermatitis. Skin cancers include melanoma, basal cell carcinoma,and squamous cell carcinoma.

Some skin disorders present various symptoms including redness,flushing, burning, scaling, pimples, papules, pustules, comedones,macules, nodules, vesicles, blisters, telangiectasia, spider veins,sores, surface irritations or pain, itching, inflammation, red, purple,or blue patches or discolorations, moles, and/or tumors. Accordingly,the biophotonic compositions and methods of the present disclosure canbe used to treat redness, flushing, burning, scaling, pimples, papules,pustules, comedones, macules, nodules, vesicles, blisters,telangiectasia, spider veins, sores, surface irritations or pain,itching, acute inflammation, red, purple, or blue patches ordiscolorations, moles, and/or tumors. Acute inflammation can presentitself as pain, heat, redness, swelling and loss of function. Itincludes those seen in allergic reactions e.g.; such as insect bites(mosquito, bees, wasps, ants, spiders etc), reaction to poison ivy orstinging nettle or the like, post-ablative treatment.

The composition may be soaked into or applied to a woven or non-wovenmaterial or a sponge and applied as a mask to body parts to treat skindisorders. A light source, such as LEDs or waveguides, may be providedwithin or adjacent the mask or the composition to illuminate thecomposition. The waveguides can be optical fibres which can transmitlight, not only from their ends, but also from their body. For example,the waveguides can be made of polycarbonate or polymethylmethacrylate.

(9) Kits

The present disclosure also provides kits for preparing and/or applyingany of the compositions of the present disclosure. The kit may include abiophotonic topical composition, as defined above, together with one ormore of a light source, devices for applying or removing thecomposition, instructions of use for the composition and/or lightsource. In some embodiments, the composition comprises at least a firstchromophore in a gelling agent. The chromophore may be present in anamount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%,7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%,25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weight of thecomposition. In embodiments where the composition comprises more thanone chromophore, the first chromophore may be present in an amount ofabout 0.01-40% per weight of the composition, and a second chromophoremay be present in an amount of about 0.0001-40% per weight of thecomposition. In certain embodiments, the first chromophore is present inan amount of about 0.01-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%,7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%,25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weight of thecomposition. In certain embodiments, the second chromophore is presentin an amount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%,5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%,22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weightof the composition. In certain embodiments, the amount of chromophore orcombination of chromophores may be in the amount of about 0.05-40.05%per weight of the composition. In certain embodiments, the amount ofchromophore or combination of chromophores may be in the amount of about0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%,12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%,30-35%, 32.5-37.5%, or 35-40.05% per weight of the composition. Thecomposition may include an oxygen-releasing agent present in amountabout 0.01%-40%, 0.01%-1.0%, 0.5%-10.0%, 5%-15%, 10%-20%, 15%-25%,20%-30%, 15.0%-25%, 20%-30%, 25%-35%, or 30%-40% by weight to weight ofthe composition. Alternatively, the kit may include the oxygen-releasingagent as a separate component to the chromophore containing composition.

In some embodiments, the kit includes more than one composition, forexample, a first and a second composition. The first composition mayinclude the oxygen-releasing agent and the second composition mayinclude the first chromophore in the gelling agent. The firstchromophore may have an emission wavelength between about 400 nm andabout 570 nm. The oxygen-releasing agent may be present in the firstcomposition in an amount of about 0.01% -1.0%, 0.5%-10.0%, 5%-15%,10%-20%, 15%-25%, 20%-30%, 15.0%-25%, 20% -30%, 25%-35%, 30%-40% or35%-45% by weight to weight of the first composition. The chromophoremay be present in the second composition in an amount of about0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%,12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%,30-35%, 32.5-37.5%, or 35-40% per weight of the second composition. Inembodiments where the second composition comprises more than onechromophore, the first chromophore may be present in an amount of about0.01-40% per weight of the second composition, and a second chromophoremay be present in an amount of about 0.0001-40% per weight of the secondcomposition. In certain embodiments, the first chromophore is present inan amount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%,7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%,25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weight of thesecond composition. In certain embodiments, the second chromophore ispresent in an amount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%,2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%,20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40%per weight of the second composition. In certain embodiments, the amountof chromophore or combination of chromophores may be in the amount ofabout 0.05-40.05% per weight of the second composition. In certainembodiments, the amount of chromophore or combination of chromophoresmay be in the amount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%,2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%,20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40.05%per weight of the second chromophore.

In some other embodiments, the first composition may comprise the firstchromophore in a liquid or as a powder, and the second composition maycomprise a gelling composition for thickening the first composition. Theoxygen-releasing agent may be contained in the second composition or ina third composition in the kit. In some embodiments, the kit includescontainers comprising the compositions of the present disclosure. Insome embodiments, the kit includes a first container comprising a firstcomposition that includes the oxygen-releasing agent, and a secondcontainer comprising a second composition that includes at least onechromophore. The containers may be light impermeable, air-tight and/orleak resistant. Exemplary containers include, but are not limited to,syringes, vials, or pouches. The first and second compositions may beincluded within the same container but separated from one another untila user mixes the compositions. For example, the container may be adual-chamber syringe where the contents of the chambers mix on expulsionof the compositions from the chambers. In another example, the pouch mayinclude two chambers separated by a frangible membrane. In anotherexample, one component may be contained in a syringe and injectable intoa container comprising the second component.

The biophotonic composition may also be provided in a containercomprising one or more chambers for holding one or more components ofthe biophotonic composition, and an outlet in communication with the oneor more chambers for discharging the biophotonic composition from thecontainer. In one embodiment, discharging the biophotonic compositionscauses the components of the composition to mix to form a biophotoniccomposition which has less than 15% leaching properties.

In other embodiments, the kit comprises a systemic or topical drug foraugmenting the treatment of the composition. For example, the kit mayinclude a systemic or topical antibiotic or hormone treatment for acnetreatment or wound healing.

Written instructions on how to use the biophotonic composition inaccordance with the present disclosure may be included in the kit, ormay be included on or associated with the containers comprising thecompositions of the present disclosure.

In certain embodiments, the kit may comprise a further component whichis a dressing. The dressing may be a porous or semi-porous structure forreceiving the biophotonic composition. The dressing may comprise wovenor non-woven fibrous materials.

In certain embodiments of the kit, the kit may further comprise a lightsource such as a portable light with a wavelength appropriate toactivate the chromophore in the biophotonic composition. The portablelight may be battery operated or re-chargeable.

In certain embodiments, the kit may further comprise one or morewaveguides.

Identification of equivalent compositions, methods and kits are wellwithin the skill of the ordinary practitioner and would require no morethan routine experimentation, in light of the teachings of the presentdisclosure. Practice of the disclosure will be still more fullyunderstood from the following examples, which are presented herein forillustration only and should not be construed as limiting the disclosurein any way.

EXAMPLES

The examples below are given so as to illustrate the practice of variousembodiments of the present disclosure. They are not intended to limit ordefine the entire scope of this disclosure.

Example 1

The photodynamic properties of (i) Fluorescein sodium salt at about 0.09mg/mL, (ii) Eosin Y at about 0.305 mg/mL, and (iii) a mixture ofFluorescein sodium salt at about 0.09 mg/mL and Eosin Y at about 0.305mg/mL in a gel according to an embodiment of the present disclosure(comprising about 12% carbamide peroxide), were evaluated. A flexstation384 II spectrometer was used with the following parameters: modefluorescence, excitation 460 nm, emission spectra 465-750 nm. Theabsorption and emission spectra are shown in FIGS. 6a and 6b whichindicate an energy transfer between the chromophores in the combination.

Example 2

The photodynamic properties of (i) Fluorescein sodium salt at 0.18 mg/mLfinal concentration, (ii) Eosin Y at about 0.305 mg/mL, and (iii) amixture of Fluorescein sodium salt at about 0.18 mg/mL and Eosin Y atabout 0.305 mg/mL in an aqueous solution were evaluated. A flexstation384 II spectrometer was used with the following parameters: modefluorescence, excitation 460 nm, emission spectra 465-750 nm. Theabsorption and emission spectra are shown in FIGS. 7a and 7b whichindicate an energy transfer between the chromophores in the combination.

Example 3

The photodynamic properties of (i) Rose Bengal at about 0.085 mg/mL,(ii) Fluorescein sodium salt at about 0.44 mg/mL final concentration,(iii) Eosin Y at about 0.305 mg/mL, and (iv) a mixture of (i), (ii) and(iii) in a gel comprising about 12% carbamide peroxide (Set A),according to an embodiment of the invention, were evaluated. Aflexstation 384 II spectrometer was used with the following parameters:mode fluorescence, excitation 460 nm, emission spectra 465-750 nm. Theabsorbance and emission spectra are shown in FIGS. 8a and 8b whichindicate an energy transfer between the chromophores in the chromophorecombination.

Example 4

The photodynamic properties of (i) Rose Bengal at about 0.085 mg/mL,(ii) Fluorescein sodium salt at about 0.44 mg/mL final concentration,(iii) Eosin Y at about 0.305 mg/mL, and (iv) a mixture of (i), (ii) and(iii) in an aqueous solution (Set A), were evaluated. A flexstation 384II spectrometer was used with the following parameters: modefluorescence, excitation 460 nm, emission spectra 465-750 nm. Theabsorbance and emission spectra are shown in FIGS. 9a and 9b whichindicate an energy transfer between the chromophores in the chromophorecombination, in the absence of an oxygen-releasing agent.

Energy transfer was also seen between: Eosin Y and Rose Bengal; PhloxineB and Eosin Y; Phloxine B, EosinY and Fluorescein, amongst othercombinations. It is to be reasonably inferred that energy transfer canalso occur in biophotonic compositions of the present disclosure.

Example 5

A randomized, split-face clinical trial of 12 weeks was performed on 90patients (ages 14-30) having moderate to severe facial acne. Moderatefacial acne was defined as having “an Investigator's Global Assessment(IGA) of 3 with 20-40 inflammatory lesions and no more than 1 nodule”.Severe facial acne was defined as having “an IGA of 4 with more than 40inflammatory lesions with the presence of more than 2 nodules and/orpresence of severe erythema and inflammatory scarring type lesion”. Foreach patient, one randomly selected side of the face was treated twice aweek for 6 weeks with a spreadable and translucent biophotoniccomposition comprising a fluorophore (Eosin Y) in a carbomerpolymer-based gel including carbamide peroxide. The biophotonic geldemonstrated less than 15% leaching of the chromophore when testedaccording to Example 6 for up to 30 minutes. The treatment comprisedtopically applying the biophotonic composition to the treatment area andexposing the composition to light from an LED light source (peakwavelength range 400-470 nm) for about 5 minutes. Other hemifaceremained untreated for the 6 week period. Both the treated and untreatedsides of the face were evaluated after 12 weeks. Results are presentedin Tables 1-5 below. The fluorescence spectra observed when thebiophotonic gel was illuminated during treatment is illustrated in FIG.10. The treatment was well tolerated by the patients and there were noserious adverse events. 80% of patients completed the study with noadverse events reported. At week 4, there was a 30% reduction ininflammatory lesions (including papules, pustules and nodules) for thetreated group compared to 9.0% reduction for untreated. At week 6, thereduction was 46.8% for treated and 18.4% for untreated, and at week 12,the reduction was 59.2% for treated and 35.6% for untreated.

TABLE 1 Total reduction from baseline at weeks 2, 4, 6, 8, 10 and 12 of≥2 IGA grades for treated and untreated hemifaces. Reduction frombaseline in IGA of ≥2 grades (%) Treated (n = 89) Untreated (n = 89)Week 2 0 (0%)  0 (0%)  Week 4 6 (6.7%) 1 (1.1%) Week 6 26 (29.2%) 6(6.7%) Week 8 28 (31.5%)  9 (10.1%) Week 10 32 (36.0%) 10 (11.2%) Week12  46 (51.7%)*  16 (18.0%)* *P < 0.0001

TABLE 2 Total reduction from baseline at weeks 2, 4, 6, 8, 10 and 12 of≥1 IGA grade for treated and untreated hemifaces. Reduction frombaseline in IGA of ≥1 grade (%) Treated (n = 89) Untreated (n = 89) Week2  9 (10.1%) 5 (5.6%) Week 4 49 (55.1%) 29 (32.6%) Week 6* 71 (79.8%) 40(44.9%) Week 8 73 (82.0%) 43 (48.3%) Week 10 76 (85.4%) 47 (52.8%) Week12+ 79 (88.8%) 62 (69.7%) *P value < 0.0001; +P value < 0.0001

TABLE 3 Total reduction from baseline at weeks 2, 4, 6, 8, 10 and 12 toIGA grade 0 or 1 for treated and untreated hemifaces. Reduction frombaseline in IGA to grade 0 or 1 (%) Treated (n = 89) Untreated (n = 89)Week 2 0 (0%)  0 (0%)  Week 4 4 (4.5%) 1 (1.1%) Week 6+ 16 (18.0%) 6(6.7%) Week 8 16 (18.0%) 8 (9.0%) Week 10 18 (20.2%)  9 (10.1%) Week 12*29 (32.6%) 10 (11.2%) +P value = 0.0213; *P value < 0.0001

TABLE 4 Proportion of patients showing at least 40% reduction frombaseline in inflammatory lesion count (includes papules, pustules andnodules) at weeks 6 and 12 for treated and untreated hemifaces. Totalreduction from baseline in inflammatory lesion count at week 12 Treated(n = 87) Untreated (n = 87) Week 6+ 56 (64.4%) 27 (31.0%) Week 12*71 71(81.6%) 40 (46.0%) +P value < 0.0001 *P value < 0.0001

TABLE 5 Summary of inflammatory lesion count and absolute changes byhemiface. Difference Treated Untreated (Treated − Untreated)Inflammatory Lesion Change Lesion Change Lesion Count* Count (%) CountChange (%) (Absolute) Change (%) Baseline n 90 90 90 Mean 23.0 23.3 −0.3(SD) (13.79) (15.41) (7.10) Week 6 n 87 87 87 87 87 87 Mean 12.4 −45.319.0 −18.6 −6.6 −26.7 (SD) (8.35) (25.15) (13.92) (33.51) (8.34) (33.86)p <0.0001 <0.0001 value Week 12 n 87 87 87 87 87 87 Mean 9.5 −55.6 15.0−32.0 −5.5 −23.6 (SD) (7.10) (32.36) (11.33) (36.61) (7.37) (37.55) p<0.0001 <0.0001 value *Includes papules, pustules and nodules

FIG. 10 is an emission spectrum showing the intensity over time of thelight being emitted from the biophotonic composition.

Example 6 Leaching Test Using Polycarbonate Membrane

FIG. 5 depicts an experimental setup of an in vitro release test forevaluating leaching of the chromophore(s) or other components (e.g.,oxygen releasing agents) from the biophotonic compositions of thepresent disclosure. In this in vitro test, a 2 mm thick layer of thebiophotonic composition is applied on an upper surface of a circularpolycarbonate (PC) membrane with a diameter of 2.4 to 3 cm, a thicknessof 10 microns, and a pore size of 3 microns. The underside of themembrane is in direct contact with phosphate saline buffer (PBS)contained in a closed compartment (i.e., the receptor compartment).Samples (100 μl×2) are then taken from the receptor compartment atdifferent time points (e.g., at 5, 10, 20, and 30 min), and evaluatedfor concentration of the chromophore(s) or any other components of thebiophotonic composition using spectrophotometry or any other suitablemethod.

For example, when the chromophore being tested is eosin, a wavelength ofabout 517 nm (absorbance) may be used. The concentration of thechromophore may then be calculated based on the chromophore standards ofknown concentration prepared in PBS and measured at the same time. Thepresence of peroxide (i.e., an indicator of the oxygen releasing agents)can also be assessed using peroxide test sticks (e.g. Quantofix Peroxide25, Sigma Aldrich).

Table 6 summarizes leaching data for different biophotonic compositionsaccording to the present disclosure. All compositions were spreadable,translucent gels, having a viscosity of about 10,000-80,000 cP. Theamount of hydrogen peroxide found in the receptor compartment was verylow for all compositions containing peroxide in Table 6. The detectionmethod of chromophore by spectrophotometry can measure the chromophoreconcentration from 0.2 μg/ml. For all biophotonic compositions tested,the release of chromophores increased over time. For all compositions,there was less than 15% chromophore leaching after 5 minutes, 10minutes, 15 minutes, and 25 minutes of incubation. All testedcompositions, other than Eosin Y (0.2%) in a carbopol polymer gelincluding urea peroxide had less than 15% chromophore leaching evenafter 30 minutes incubation, which is longer than a treatment timeaccording to many embodiments of the present disclosure.

The effect of illumination on chromophore leaching from the biophotoniccompositions was also investigated. It was found that illumination ofthe biophotonic compositions with light for 5 minutes at a distance of 5cm induced photobleaching of the chormophore(s). In fact, thechomophores photobleached in about 2-3 minutes. In these cases, thechromophore(s) were undetectable in the receptor compartment. Therefore,during treatment involving light illumination, even lower chromophoreleaching than the results presented in Table 9 can be reasonablyexpected.

TABLE 6 Percentage of chromophores released from biophotoniccompositions according to embodiments of the present disclosure, withtime of incubation. Percentage chromophore released into receptorcompartment from composition with time of incubation (n = 3) 5 10 20 30Composition mins mins mins mins Eosin Y (0.011%), carbopol gel Not Not0.75 0.78 (1.7%), urea peroxide gel (12%), detect- detect- saffron,glycerine, propylene able able glycol, hyaluronic acid. Fluorescein(0.2%), carbopol gel 2.71 4.85 4.72 4.84 (1.7%), urea peroxide (12%),saffron, glycerine, propylene glycol, hyaluronic acid. Rose Bengal(0.2%), carbopol gel 2.39 3.32 5.26 5.21 (1.7%), urea peroxide (12%),saffron, glycerine, propylene glycol, hyaluronic acid. Rose Bengal(0.1%) + Fluorescein 2.91 5.21 8.48 8.43 (0.1%), carbopol gel (1.7%),urea peroxide gel (12%), saffron, glycerine, propylene glycol,hyaluronic acid. Phloxin B (0.2%), carbopol gel 0.54 2.39 4.62 4.50(1.7%), urea peroxide gel (12%), saffron, glycerine, propylene glycol,hyaluronic acid. Eosin Y (0.2%), carbopol gel 2.77 2.72 6.56 9.08(1.7%), urea peroxide gel (12%), saffron, glycerine, propylene glycol,hyaluronic acid. Phloxin B (0.1%) + Fluorescein 2.28 4.49 7.56 11.02(0.1%), carbopol gel (1.7%), urea peroxide gel (12%), saffron,glycerine, propylene glycol, hyaluronic acid. Phloxin B (0.1%) + RoseBengal 2.41 2.36 5.14 4.90 (0.1%), carbopol gel (1.7%), urea peroxidegel (12%), saffron, glycerine, propylene glycol, hyaluronic acid.Phloxin B (0.1%) + Eosin Y 3.84 6.25 10.08 12.00 (0.1%) carbopol gel(1.7%), urea peroxide (12%), saffron, glycerine, propylene glycol,hyaluronic acid. Rose Bengal (0.1%) + Eosin Y 3.04 4.28 6.63 8.12(0.1%), carbopol gel (1.7%), urea peroxide (12%), saffron, glycerine,propylene glycol, hyaluronic acid. Fluorescein (0.1%) + Eosin Y 2.963.99 5.78 7.58 (0.1%), carbopol gel (1.7%), urea peroxide (12%),saffron, glycerine, propylene glycol, hyaluronic acid. Phloxin B(0.1%) + Eosin Y 1.00 2.3 4.48 5.80 (0.1%), carbopol gel Eosin Y (0.2%),carbopol 6.78 8.2 14.38 17.89 gel (1.7%), urea peroxide gel (12%) EosinY (0.2%), carbopol 3.34 4.90 7.30 9.26 gel (1.7%), saffron, glycerine,propylene glycol Phloxin B (0.1%) + Eosin Y 0.51 0.25 1.79 3.14 (0.1%),gelatin gel (5%) Rose Bengal (0.1%) + Eosin Y 0 0.39 1.39 2.15 (0.1%),gelatin gel (5%) Eosin Y (0.2%), starch gel (8%) 2.91 3.72 7.11 9.06Eosin Y (0.2%), sodium 3.41 6.24 9.93 12.77 hyaluronate gel (2%)

Example 7 Angiogenic Potential of the Biophotonic Composition of theDisclosure

A human skin model was developed to assess the angiogenic potential ofthe biophotonic composition of the present disclosure. Briefly, abiophotonic composition comprising fluorophores (Eosin Y andErythrosine) in a carbomer polymer-based gel including urea peroxide,was placed on top of a human skin model containing fibroblasts andkeratinocytes. The spreadable and translucent biophotonic compositionhad less than 15% leaching of chromophore for up to 30 minutes, whentested separately according to Example 6. The skin model and thecomposition were separated by a nylon mesh of 20 micron pore size. Thecomposition was then irradiated with blue light (‘activating light’) for5 minutes at a distance of 5 cm from the light source. The activatinglight consisted of light emitted from an LED lamp having an average peakwavelength of about 400-470 nm, and a power intensity measured at 10 cmof 7.7 J/cm² to 11.5 J/cm². Upon illumination with the activating light,the biophotonic composition emitted fluorescent light (FIG. 4). Sincethe biophotonic composition was in limited contact with the cells, thefibroblasts and keratinocytes were exposed mainly to the activatinglight and the fluorescent light emitted from the biophotoniccomposition. Conditioned media from the treated human 3D skin model werethen applied to human aortic endothelial cells previously plated inmatrigel. The formation of tubes by endothelial cells was observed andmonitored by microscopy after 24 hours. The conditioned medium from 3Dskin models treated with light illumination induced endothelial tubeformation in vitro, suggesting an indirect effect of the light treatment(blue light and fluorescence) on angiogenesis via the production offactors by fibroblasts and keratinocytes. Plain medium and conditionedmedium from untreated skin samples were used as a control, and did notinduce endothelial tube formation.

FIG. 11 is an emission spectrum showing the intensity over time of thelight being emitted from the biophotonic composition.

Example 8 Protein Secretion and Gene Expression Profiles

Wounded and unwounded 3D human skin models (EpiDermFT, MatTekCorporation) were used to assess the potential of a biophotoniccomposition of the present disclosure to trigger distinct proteinsecretion and gene expression profiles. Briefly, a biophotoniccomposition comprising Eosin and Erythrosine in a carbomer polymer-basedgel including urea peroxide, was placed on top of wounded and unwounded3D human skin models cultured under different conditions (with growthfactors, 50% growth factors and no growth factors). The spreadable andtranslucent biophotonic gel had less than 15% leaching of chromophoreduring a 30 minute test time, according to Example 6. The skin modelsand the composition were separated by a nylon mesh of 20 micron poresize. Each skin model-composition combination was then irradiated withblue light (‘activating light’) for 5 minutes at a distance of 5 cm fromthe light source. The activating light consisted of light emitted froman LED lamp having an average peak wavelength of about 440-470 nm, apower density of 60-150 mW/cm² at 5 cm, and a total intensity after 5minutes of about 18-39 J/cm². The controls consisted of 3D skin modelsnot illuminated with light.

Gene expression and protein secretion profiles were measured 24 hourspost-light exposure. Cytokine secretion was analyzed by antibody arrays(RayBio Human Cytokine antibody array), gene expression was analyzed byPCR array (PAHS-013A, SABioscience) and cytotoxicity was determined byGAPDH and LDH release. Results (Tables 7 and 8) showed that the lighttreatment is capable of increasing the level of protein secreted andgene expression involved in the early inflammatory phase of woundhealing in wounded skin inserts and in non-starvation conditions. Instarvation conditions mimicking chronic wounds, there was no increase inthe level of inflammatory protein secreted when compared to the control.Interestingly, the effect of the light treatment on unwounded skinmodels has a much lower impact at the cellular level than on woundedskin insert, which suggests an effect at the cellular effect level ofthe light treatment. It seems to accelerate the inflammatory phase ofthe wound healing process. Due to the lack of other cell types such asmacrophages in the 3D skin model, the anti-inflammatory feed-back isabsent and may explain the delay in wound closure. Cytotoxicity was notobserved in the light treatments.

TABLE 7 List of proteins with statistically significant differencesecretion ratio between treated and untreated control at day 3. Twoarrows mean that the ratio was over 2 folds. Medium 1X Medium 0.5XMedium 0X Increase ENA78 p = 0.04 ↑↑ Angiogenin p = 0.03 ↑ Il-1R4/ST2 p= 0.02 ↑↑ CXCL16 p = 0.04 ↑ MMP3 p = 0.01 ↑↑ MCP-2 p = 0.04 ↑↑ DecreaseBMP6 p = 0.01 ↓ BMP6 p = 0.02 ↓ TNFα p = 0.005 ↓

TABLE 8 List of genes with statistically significant differenceexpression ratio between treated and untreated control during the first24 hours. Two arrows mean that the ratio was over 2 folds. Medium 1XMedium 0.5X Medium 0X Increase CTGF p = 0.02 ↑ CTGF P = 0.04 ↑ MMP3 p =0.007 ↑↑ ITGB3 p = 0.03 ↑ ITGB3 p = 0.05 ↑ LAMA1 p = 0.03 ↑ MMP1 p =0.03 ↑ MMP1 p = 0.02 ↑↑ ITGA2 p = 0.03 ↑ MMP3 p = 0.01 ↑ MMP10 p = 0.003↑↑ THBS1 P = 0.02 ↑ MMP3 p = 0.007 ↑↑ MMP8 p = 0.02 ↑↑ THBS1 p = 0.03 ↑Decrease HAS1 p = 0.009 ↓↓ NCAM1 p = 0.02 ↓↓ NCAM1 p = 0.05 ↓↓ VCAN p =0.02 ↓ VCAM1 p = 0.03 ↓↓ LAMC1 p = 0.002 ↓ COL7A1 p = 0.04 ↓ COL6A1 p =0.007 ↓ CTNNA1 p = 0.03 ↓ MMP7 p = 0.003 ↓

Example 9 Collagen Formation in Skin

A randomized, placebo-controlled, single-blinded, split face and singlehand study of 32 patients, split into 4 groups (A, B, C and D), assessedthe safety and efficacy of treatment once a week for 4 weeks: (A) “lightalone”—light, according to an embodiment of the present disclosure,comprising light from an LED source having an average peak wavelength ofabout 400-490 nm at a power density of less than 150 mW/cm² for 5minutes; and a placebo formulation; (B) “light+gel”—light as in (A) plusbiophotonic gel according to an embodiment of the present disclosure);(C) “gel alone”—biophotonic gel as in (B) and a sham light (white LEDlight); and (D) 0.1% retinoic based cream. The biophotonic gel includeda fluorophore in a carbopol gel and urea peroxide, the gel having aviscosity of about 10,000 cP to 50,000 cP, and demonstrating less than15% leaching when tested for up to 30 minutes according to Example 6.The gel was translucent and spreadable. Skin biopsies were obtainedbefore treatment and 12 weeks after treatment from the treatment site.Histological samples of the skin biopsies were graded by an independentand experienced pathologist blinded to the treatment assignment. Theresults are presented in Table 9 below and show that the light treatmentwith and without the biophotonic gel, according to embodiments of thepresent disclosure, showed a 287% and 400% increase from the baseline,respectively, in collagen clusters as viewed through Gomori Trichomestaining, in the treated areas of skin. There were no serious adverseevents. There was no reported or observed photosensitivity, inflammationor pain.

TABLE 9 Semi-quantitative histological collagen evaluation % increaseTreatment in collagen Photoactivatable composition excited with light400 having 460 nm peak wavelength Placebo composition + light having 460nm peak 287 wavelength Retinol cream with no light 189 Placebocomposition with white light 150

Example 10 Flap Closure

A caudally based rectangular flap was elevated in the back of Wistarrats. A silicone sheet was inserted beneath the skin flap to preventadhesion and reperfusion of the flap from the underlying tissues.Following flap closure, a biophotonic gel according to an embodiment ofthe present disclosure was applied onto the dorsal flap in a thinmonolayer (2 mm) and exposed to a light, for 5 minutes, from a LED lightsource having a peak wavelength of about 440-470 nm. The spreadablebiophotonic gel included a fluorophore in a carbopol gel and ureaperoxide, the gel having a viscosity of about 10,000 cP to 50,000 cP,and demonstrating less than 15% leaching when tested for up to 30minutes according to Example 6. The biophotonic gel was removed and skinspecimens were collected from different areas in the flap forhistological analyses nine days post-treatment. The treated groupdemonstrated a significantly greater number of Ki67-positive-stainingevents (P=0.02) compared to those in the non-treated group theseresults, suggesting that the treatment may modulate the proliferation ofthe cells involved in wound healing (FIG. 12). Following examination byan external pathologist, the treatment group was associated with asignificant (P<0.05) decrease in the coagulative necrosis in theepidermis and an increase of the fibrillar stroma (dermis) as comparedto the control group.

Example 11 Evaluation of Removal of Biophotonic Composition from EthanolSoaked Paper

Regular white print paper was soaked in 70% ethanol (EtOH). A 2 mmthickness of different embodiment's of biophotonic compositionsaccording to the present disclosure (Table 10) were placed onto thesoaked paper and left for 5 minutes. After 5 minutes, the compositionswere washed off with 70% EtOH. A composition comprising Eosin (0.017%),silica particles, modified starch, and hydrogen peroxide was alsotested.

The results show that biophotonic compositions of the present disclosureincluding a carbamide gel do not stain white paper. A compositioncontaining Eosin and another hydrophilic polymer (starch) in combinationwith silica particles did stain the paper.

TABLE 10 Evaluation of removal of biophotonic composition from paperBiophotonic composition Colour of paper after washing Eosin (0.017%),silica particles, Orange/red stain on paper modified starch, hydrogenperoxide observed. (included for comparison only). Eosin (0.011%) in aurea peroxide, Substantially white - no glycerin, propylene glycol,carbopol, staining observed. hyaluronic acid, glucosamine gel. Eosin(0.011%) + carbamide Substantially white - no peroxide + 1.8% carbopol940 staining observed.

Example 12 Evaluation of Heat Dissipation During Illumination of aBiophotonic Composition

A 3 mm thick layer of a biophotonic composition according to anembodiment of the present disclosure comprising a fluorescentchromophore in a carbopol gel according to an embodiment of the presentdisclosure was applied on the skin of hands of volunteers with differentskin types and illuminated for 5 minutes with a blue LED light having apower density of about 50 to 150 mW/cm² at a distance of 5 cm from thelight. The biophotonic gel was spreadable and had less than 15% byweight leaching when tested according to Example 6. A thermometer probewas placed within the composition, at the surface of the skin, and thetemperature was monitored in real-time during illumination of thecomposition. The skin temperature with no composition but the same lightillumination was also measured for the same volunteers. The skin typestested were, according to Fitzpatrick classification scales, type III(white skin, sometimes burns and gradually tans), type IV (beige tobrown skin, rarely burns and easily tans) and type VI (black skin, neverburns, very easily tans). The results are shown in table 11.

TABLE 11 Temperature of skin under biophotonic composition duringillumination for 5 minutes compared to temperature skin with nocomposition and illumination alone Minimum-maximum Minimum-maximumtemperature of skin temperature of skin under composition during withoutcomposition during 5 mins of illumination/° C. 5 mins. of illumination/°C. (Average over 5 mins/° C.) (Average over 5 mins/° C.) Skin Type III26.5-35.1 (32.2) 28.7-39.1 (36.2) Skin Type IV 27.6-39.9 (36.1)31.4-39.9 (37.0) Skin Type VI 28.5-39.9 (35.6) 29.6-40.0 (37.4)

All skin types with biophotonic composition applied demonstrated aslower temperature increase compared to bare skin (no biophotoniccomposition), and so the biophotonic composition conferred a buffereffect. After 5 minutes of light illumination, the temperature of theskin under the biophotonic composition for all volunteers reached amaximum of 39.9° C., compared to 40° C. with light alone and bare skin.Overall no pain, burning or discomfort was felt by the volunteers.

Example 13 Selecting the Concentration of Chromophore in the BiophotonicComposition

The fluorescence spectra of biophotonic compositions with differentconcentrations of chromophores were investigated using aspectrophotometer and an activating blue light. Exemplary fluorescencespectra of Eosin Y and Fluorescein are presented in FIG. 13. It wasfound that emitted fluorescence from the chromophore increases rapidlywith increasing concentration but slows down to a plateau with furtherconcentration increase. Activating light passing through the compositiondecreases with increasing chromophore composition as more is absorbed bythe chromophores. Therefore, the concentration of chromophores inbiophotonic compositions of the present disclosure can be selectedaccording to a required ratio and level of activating light andfluorescence treating the tissue based on this example. In someembodiments, it will be after the zone of rapid increase, i.e. between0.5 and 1 mg/mL for Eosin Y (FIG. 13).

Therefore, concentration can be selected according to requiredactivating light and fluorescence. In some embodiments, it will be afterthe zone of rapid increase, i.e. between 0.5 and 1 mg/mL for Eosin Y(FIG. 13). A person skilled in the art would also take into account theeffect on fluorescence of other ingredients in a composition and adaptthe concentration of the chromophore accordingly. For example, certaingelling agents bind to certain chromophores which may lower theirfluorescence. One example is albumin. In such cases, a higherconcentration of the chromophore can be used in the composition.

Example 14 Eosin and Rose Bengal Act in a Synergistic Manner

The synergy between two chromophores according to various embodiments ofthe present disclosure was investigated by preparing the following:

1—Eosin Y (0.035%)+Rose Bengal (0.085%) in a 12% carbamide gel.

2—Rose Bengal (0.085%) in a 12% carbamide gel

Rose Bengal is known to have a high quantum yield in terms of oxygenproduction in the presence of oxygen-releasing agents whenphotoactivated by green light. Eosin Y is known to have a high quantumyield in terms of emitted fluorescent light when photoactivated and canbe at least partially activated by blue light when in a gel.Photoactivated Eosin Y does not have a high quantum yield in terms ofoxygen production in the presence of oxygen-releasing agents. When EosinY and Rose Bengal are combined, it appears that both chromophores areactivated by the same blue light as evidenced by FIG. 14.

FIG. 14, left panel, shows a photograph of the composition when viewedunder a light microscope (×250) before exposure to an activating light.Very few bubbles were seen in both compositions. Following illuminationwith blue light, right panel, a dramatic increase in bubbles was seenwith the composition comprising a combination of Eosin Y and RoseBengal, but not with the composition comprising Rose Bengal alone. Thissuggests that there is a transfer of energy from Eosin Y to Rose Bengalleading to the formation of oxygen species.

Example 15 Viscosity of Spreadable Compositions

Gels, based on carbopol polymers with differing concentrations, wereevaluated for their suitability for use in embodiments of biophotoniccompositions of the present disclosure. The viscosity, spreadability andability to stay in place on tissue of the gels were evaluated. The gelscomprised carbopol 940, glycerin, propylene glycol, water, as well assmall amounts of a chelator, a pH adjuster, healing factors andpreservatives. Ten gel compositions were tested having varying amountsof carbopol 940, all other ingredient concentrations remaining the same.Viscosity was evaluated using (1) Brookfield DV-II+Pro viscometer:spindle 7, 50 rpm, 1 minute; and (2) Brookfield HN viscometer: spindleCP51, 2 rpm. Ability to be easily spread was evaluated based on ease offorming a 2 mm thick layer on a surface and ability to conform to thesurface topography. Ability to stay in place was evaluated by placing a2 mm thick layer of each gel on a surface of a pork chop having an 8 mmdiameter biopsy punch in order to simulate a wound. The steak was thenpositioned such that the surface having the gel thereon was at about 90°to a horizontal plane (i.e. the gel was substantially vertical) and wasthen left in place for 5 minutes at room temperature. The results aresummarized in Table 12.

TABLE 12 Evaluation of viscosity, spreadability and ability to stay inplace of gels having different carbopol concentrations. Vis- Vis- % bywt cosity cosity Ability to Ability to Gel Carbopol (cP) (1) (cP) (2) bespread stay in place 1 0.2 0 0 Too liquid Too liquid 2 0.5 800 828 Tooliquid Too liquid 3 1.0 11920 11737 Good Good 4 1.7 33840 38110 GoodGood 5 2.0 71520 74563 Not easy Good to make gel conform to the woundtopography 6 2.5 74080 74770 Very difficult Good to make gel conform tothe wound topography 7 1.1 15840 15948 Good Good 8 1.3 21280 22783 GoodGood 9 1.5 31360 33346 Good Good 10 1.85 44320 49295 Good Good

Gels which did not flow when positioned vertically were obtained with acarbopol wt % of more than 0.5% and higher. Gels with a carbopol wt % ofmore than 0.5% and less than 2 wt % could be spread as a 2 mm thicklayer. These gels may be suitable gelling agents for biophotoniccompositions of the present invention. Other concentrations of carbopolmay also be used in conjunction with thickening agents or diluters inbiophotonic compositions of the present disclosure. Also, other carbopolgrades, polymers and other gelling agents may also have propertiessuitable for use as a gelling agent in the present compositions.

It should be appreciated that the invention is not limited to theparticular embodiments described and illustrated herein but includes allmodifications and variations falling within the scope of the inventionas defined in the appended claims.

The invention claimed is:
 1. A biophotonic composition comprising: afirst chromophore; wherein the first chromophore is a xanthene dyehaving an absorption wavelength of between about 200 nm and about 500nm; and a carbomer present in the biophotonic composition in an amountof between about 1.0% and about 2.0% by weight of the final composition,such that the biophotonic composition has a viscosity ranging from about15,000 cP and about 50,000 cP, and to render the biophotonic compositionsubstantially resistant to leaching such that less than 15% by weight ofthe total chromophore amount leaches out of the biophotonic composition;wherein the first chromophore is dispersed throughout the biophotoniccomposition; wherein the biophotonic composition does not comprise anoxidant; and wherein the biophotonic composition is photoactivatableafter a period of illumination of between about 1 second and about 30seconds with an actinic light delivering a power density of less thanabout 150 mW/cm² when located at 5 cm from the biophotonic composition;and when photoactivated, the biophotonic composition emits light havinga wavelength ranging from about 490 nm to about 800 nm.
 2. Thebiophotonic composition of claim 1, wherein the substantial resistanceto leaching comprises less than 15% by weight of the total chromophoreamount leaching out of the biophotonic composition into a phosphatesaline buffer solution contained in a receptor compartment, through a2.4-3 cm diameter polycarbonate (PC) membrane with a thickness of 10microns and a pore size of 3 microns, having a top side onto which a 2mm thick layer of the biophotonic composition is placed for 5 minutes atroom temperature and pressure, and a bottom side which is in directcontact with the phosphate saline buffer solution.
 3. The biophotoniccomposition of claim 1, wherein the biophotonic composition istranslucent.
 4. The biophotonic composition of claim 3, wherein thetranslucency comprises at least 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 85%, 90%, 95% or 100% transmittance of light through a 2 mmthick composition.
 5. The biophotonic composition of claim 1, furthercomprising a humectant.
 6. The biophotonic composition of claim 5,wherein the humectant is glycerine.
 7. The biophotonic composition ofclaim 1, wherein the first chromophore is selected from the groupconsisting of Eosin Y, Eosin B, Fluorescein, Rose Bengal and Phloxin B.8. The biophotonic composition of claim 1, wherein the first chromophoreis present in an amount of about 0.001% to about 40% by weight of thetotal composition.
 9. The biophotonic composition of claim 1, whereinthe biophotonic composition further comprises a second chromophore. 10.The biophotonic composition of claim 9, wherein the first chromophorehas an emission spectrum that overlaps at least 20% with an absorptionspectrum of the second chromophore.
 11. The biophotonic composition ofclaim 9, wherein the first chromophore is Eosin Y, and the secondchromophore is Fluorescein, and/or Phloxine B.
 12. The biophotoniccomposition of claim 9, wherein the first chromophore is Fluorescein,and the second chromophore is Eosin Y.
 13. The biophotonic compositionof claim 1, wherein the light emitted by the biophotonic composition hasa power density of between 0.005 to about 10 mW/cm².
 14. A method forproviding cosmetic treatment, comprising: applying topically thebiophotonic composition according to claim 1 to a skin; and illuminatingsaid biophotonic composition with light having a wavelength thatoverlaps with an absorption spectrum of the first chromophore.
 15. Amethod for biophotonic treatment of a skin disorder, comprising:applying topically the biophotonic composition according to claim 1, toa target skin tissue afflicted with the skin disorder; and illuminatingsaid biophotonic biophotonic composition with light having a wavelengththat overlaps with an absorption spectrum of the first chromophore. 16.The method of claim 15, wherein the skin disorder is acne.
 17. Themethod of claim 15, wherein the biophotonic composition is illuminatedfor about 1 minute to about 30 minutes.
 18. The method of claim 15,wherein the biophotonic composition is illuminated with visiblenon-coherent light.